A Splice Form of VEGF, a Potential Anti-Angiogenetic Form of Head and Neck Squamous Cell Cancer Inhibition

Angiogenesis, primarily mediated by vascular endothelial growth factor (VEGF), is a fundamental step in the progression and metastasis of head and neck squamous cell carcinoma (HNSCC). Traditional anti-angiogenic therapies that target the VEGF pathway have shown promise but are often associated with significant side effects and variable efficacy due to the complexity of the angiogenic signaling pathway. This review highlights the potential of a specific VEGF splice form, VEGF165b, as an innovative therapeutic target for HNSCC. VEGF165b, unlike standard VEGF, is a natural inhibitor that binds to VEGF receptors without triggering pro-angiogenic signaling. Its distinct molecular structure and behavior suggest ways to modulate angiogenesis. This concept is particularly relevant when studying HNSCC, as introducing VEGF165b’s anti-angiogenic properties offers a novel approach to understanding and potentially influencing the disease’s dynamics. The review synthesizes experimental evidence suggesting the efficacy of VEGF165b in inhibiting tumor-induced angiogenesis and provides insight into a novel therapeutic strategy that could better manage HNSCC by selectively targeting aberrant vascular growth. This approach not only provides a potential pathway for more targeted and effective treatment options but also opens the door to a new paradigm in anti-angiogenic therapy with the possibility of reduced systemic toxicity. Our investigation is reshaping the future of HNSCC treatment by setting the stage for future research on VEGF splice variants as a tool for personalized medicine.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with approximately 600,000 new cases diagnosed annually.The mortality rate remains significant, with a five-year survival rate of around 50%, largely due to latestage diagnosis and the aggressive nature of the disease.Major risk factors contributing to the development of HNSCC include tobacco use, excessive alcohol consumption, and infection with human papillomavirus (HPV).Given the high incidence and mortality rates, there is an urgent need for more effective therapeutic strategies, including those targeting angiogenesis [1].
Angiogenesis, the process of new blood vessel formation, is a critical event in the progression and metastasis of various cancers, including HNSCC [2].Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, promoting endothelial cell proliferation, migration, and vascular permeability.The discovery of VEGF and its role in angiogenesis revolutionized cancer research, leading to the development of anti-angiogenic therapies targeting VEGF pathways [3].
Despite advances in anti-angiogenic therapy, challenges remain in accurately assessing the efficacy of these treatments due to the lack of standardized criteria [4].Furthermore, while VEGF expression is detected by immunohistochemistry (IHC) in both normal and tumor tissues, its behavior does not always correlate with new vessel formation, suggesting a more nuanced role in the regulation of angiogenesis.The role of biomarkers in cancer diagnosis and treatment is well recognized.For example, the detection of thymine dimers in renal cell carcinoma (RCC) using immunohistochemistry highlights the importance of identifying novel biomarkers for better clinical management and therapeutic targeting in different types of cancer, including HNSCC [5][6][7][8].Interestingly, adding complexity to our understanding of VEGF's role in angiogenesis regulation, certain splice forms of VEGF exhibit inhibitory properties.Among these splice variants, VEGF165b has received attention for its ability to block the pro-angiogenic effects of other VEGF isoforms by binding to VEGF receptors [9].
The discovery of VEGF as a key regulator of angiogenesis represents a defining moment in cancer research and therapy development.VEGF was first identified in the 1980s, with significant contributions from researchers such as Napoleone Ferrara, who isolated and cloned VEGF in 1989, revealing its important role in angiogenesis [10].This discovery paved the way to understanding how tumors stimulate blood vessel formation to sustain their growth and spread, a process essential for tumor progression and metastasis [11].VEGF acts by promoting endothelial cell proliferation, migration, and increased vascular permeability.Its role in angiogenesis has rapidly positioned VEGF as a prime target for therapeutic intervention in cancer treatment.The recognition of VEGF's involvement in tumor angiogenesis has led to the development of anti-angiogenic therapies aimed at inhibiting the VEGF pathway to deprive tumors of blood supply, thus inhibiting their growth and metastatic potential [12].Another important role of VEGF is in vascular endothelial development and optimal vascular endothelial function, and it is recognized for its role in the maturation of arterial and venous endothelium.This mechanism, essential in the physiological context, also extends to pathological conditions, particularly in the well-vascularized environment of malignant tumors.Given the importance of VEGF, its inhibitors used in oncological treatments justify prudent use, especially given their potential contribution to arterial aneurysm and dissection [13,14].This phenomenon underscores the need for cardiovascular and oncology professionals to remain vigilant to this rare but serious risk, ensuring a balanced approach to VEGF inhibitor therapy in cancer patients [15].
In the evolution of cancer therapy, particularly in HNSCC, the promise of antiangiogenic therapy has opened up new ways of treatment.Despite the theoretical attractiveness and preclinical success of anti-angiogenic therapies, a significant challenge in their clinical application is the lack of standardized criteria for evaluating their efficacy.This gap not only hinders the evaluation of current treatments but also hampers the development of future anti-angiogenic strategies [16,17].
Challenges in evaluating the efficacy of anti-angiogenic therapy: 1.
Heterogeneous response: Tumors can vary greatly in their response to anti-angiogenic therapy, not only between different cancers but also between patients with the same cancer.This variability makes it difficult to establish universal criteria for efficacy and treatment [18].

2.
The dynamic nature of tumor angiogenesis: Tumors can adapt to anti-angiogenic therapy over time, either by activating alternative angiogenic pathways or by adopting less angiogenesis-dependent mechanisms.This ability to adapt means that the efficacy of therapy may decrease with time or that initial responses may not be sustained, complicating the assessment of long-term efficacy [19].

3.
Lack of direct biomarkers: There is a lack of direct, reliable biomarkers that can accurately reflect changes in angiogenesis due to therapy.Most current methods assess tumor size or growth rates using imaging techniques such as MRI (magnetic resonance imaging) or CT (computed tomography).However, these indicators may not sensitively or specifically reflect changes in angiogenesis, especially in the early stages of treatment [20].

4.
Difficulty in measuring microenvironment changes: Anti-angiogenic therapies not only affect tumor cells, but also have a significant impact on the tumor microenvi-ronment, including altering vascular permeability, interstitial pressure, and hypoxia.These changes are difficult to measure directly and quantitatively in patients [21].
The presence of VEGF in a wide range of normal and tumor tissues has been well documented by IHC, a vital technique for visualizing specific proteins in tissue sections.VEGF, as the main regulator of angiogenesis, is known to correlate with the formation of new blood vessels, which is a distinctive sign of tumor growth and metastasis.However, VEGF expression does not consistently determine angiogenesis, indicating a more complex role than simply as a direct facilitator of new vessel formation [22].
In the context of normal physiology, VEGF expression is essential for various functions, including wound healing and the menstrual cycle, where angiogenesis is a normal and controlled process.In these situations, VEGF expression is tightly regulated, and its effects are balanced by a suite of other angiogenic and anti-angiogenic factors that maintain vascular homeostasis [23].In contrast, tumor tissues often show increased VEGF expression, which has been commonly associated with the promotion of angiogenesis within the tumor microenvironment.This angiogenic change is considered an essential step in the transition of tumors from a latent to a malignant, invasive condition.Increased levels of VEGF are expected to correspond with increased angiogenesis and, consequently, tumor progression [24].
VEGF achieves its pro-angiogenic effects by binding to specific receptors on the surface of endothelial cells, which leads to a cascade of signaling events that result in the proliferation, migration, and formation of new blood vessels by endothelial cells [25].In addition to its known angiogenic role, VEGF also has naturally occurring inhibitory forms.These inhibitory forms of VEGF are splicing variants that come from the same VEGF gene but are processed differently, resulting in proteins that can inhibit the effects of the standard VEGF molecule.The most studied of these is VEGF165b, an isoform that competes with the angiogenic form of VEGF for binding to the receptor but does not activate the receptor signaling pathways that lead to angiogenesis.Instead, it inhibits these pathways, resulting in an anti-angiogenic effect [26].
Of the VEGF splice variants, VEGF165b has emerged as a molecule of interest because of its intrinsic anti-angiogenic properties.Unlike its pro-angiogenic homologs, VEGF165b does not stimulate vascular growth [27]; instead, it binds to VEGF receptors with similar affinity but without activating the angiogenic signaling cascade [28].This unique feature of VEGF165b positions it as a novel and promising target for the inhibition of angiogenesis in the context of squamous cell carcinoma of the head and neck.The therapeutic potential of VEGF165b is based on its ability to naturally inhibit vascular proliferation, which is essential for HNSCC progression and metastasis [29].Understanding this form of splicing could lead to the development of innovative anti-angiogenic strategies, potentially improving patient outcomes by preventing tumor growth and spread while minimizing the side effects associated with conventional anti-angiogenic drugs.

Understanding the Splice Forms of VEGF 2.1. The Biology of VEGF Splicing
Angiogenesis is indispensable for both physiological growth and pathological developments, such as tumorigenesis in HNSCC [17].VEGF, an essential factor in angiogenic regulation, is synthesized by a finely regulated process known as alternative splicing.This post-transcriptional mechanism facilitates the generation of multiple VEGF isoforms from a single gene, enriching the VEGF family to include both the promotion and inhibition of angiogenesis [30].
The VEGF gene is alternatively spliced through its series of exons to produce different isoforms, each with different receptor-binding characteristics and subsequent biological activities.The pro-angiogenic isoforms-VEGF121, VEGF165, and VEGF189-are capable of binding to the VEGF receptors, VEGFR1 and VEGFR2, on endothelial cells, thereby initiating a cascade of cellular events leading to proliferation, migration, and ultimately new vessel formation (Figure 1) [31].
activities.The pro-angiogenic isoforms-VEGF121, VEGF165, and VEGF189-are capable of binding to the VEGF receptors, VEGFR1 and VEGFR2, on endothelial cells, thereby initiating a cascade of cellular events leading to proliferation, migration, and ultimately new vessel formation (Figure 1) [31].These isoforms also have different affinities for heparin and heparan sulfate proteoglycans, determining their solubility and influence on the extracellular matrix, which in turn influences tissue-specific vascular growth patterns [32].
In contrast, VEGF165b, together with the b-series VEGFs, represents the antiangiogenic faction of the VEGF family.This variant results in a protein with a divergent C-terminal domain that modulates receptor interaction due to the inclusion of the alternative exon 9b during mRNA splicing.This alteration prevents normal activation of VEGFR2-mediated pathways essential for angiogenic signaling, thereby positioning VEGF165b as a competitive inhibitor of its pro-angiogenic relatives.It serves as a critical natural antagonist in the angiogenic balance by binding to VEGF receptors without initiating the angiogenic signaling cascade [33].It is noteworthy that VEGF165b binds to VEGF receptors with similar affinity but does not trigger angiogenic signaling pathways, making it a natural antagonist of the pro-angiogenic VEGF family.This unique feature positions VEGF165b as a promising target for anti-angiogenic therapy, particularly in diseases where angiogenesis plays a crucial role, such as HNSCC (Table 1) [27].These isoforms also have different affinities for heparin and heparan sulfate proteoglycans, determining their solubility and influence on the extracellular matrix, which in turn influences tissue-specific vascular growth patterns [32].
In contrast, VEGF165b, together with the b-series VEGFs, represents the anti-angiogenic faction of the VEGF family.This variant results in a protein with a divergent C-terminal domain that modulates receptor interaction due to the inclusion of the alternative exon 9b during mRNA splicing.This alteration prevents normal activation of VEGFR2-mediated pathways essential for angiogenic signaling, thereby positioning VEGF165b as a competitive inhibitor of its pro-angiogenic relatives.It serves as a critical natural antagonist in the angiogenic balance by binding to VEGF receptors without initiating the angiogenic signaling cascade [33].It is noteworthy that VEGF165b binds to VEGF receptors with similar affinity but does not trigger angiogenic signaling pathways, making it a natural antagonist of the pro-angiogenic VEGF family.This unique feature positions VEGF165b as a promising target for anti-angiogenic therapy, particularly in diseases where angiogenesis plays a crucial role, such as HNSCC (Table 1) [27].

Inhibits angiogenic pathways, acts as a competitive inhibitor
Anti-angiogenic In addition to VEGF165b, the VEGF family includes several other isoforms, such as VEGF121b, which also exhibit anti-angiogenic properties.VEGF121b, like VEGF165b, is a splice variant of VEGF-A that plays a significant role in inhibiting angiogenesis and tumor progression.This variant is characterized by its shorter sequence, which lacks the heparin-binding domain, allowing it to remain more diffusible in the extracellular matrix [34].
Recent studies have shown that VEGF121b can effectively bind to VEGF receptors (VEGFR1 and VEGFR2) without triggering the pro-angiogenic signaling pathways typically activated by other VEGF-A isoforms.Bates et al. (2002) demonstrated that VEGF165b, an inhibitory splice variant of VEGF-A, binds to VEGFR2 with an affinity comparable to VEGF165 but does not induce the phosphorylation required for downstream signaling, thereby inhibiting angiogenesis in vivo.Given the structural similarities, VEGF121b is believed to function similarly and has been observed to suppress tumor growth in various cancer models, highlighting its potential as a therapeutic agent [35].
The inclusion of VEGF121b in the broader context of anti-angiogenic VEGF isoforms underscores the diversity and complexity of the VEGF signaling network.By understanding the distinct roles of these isoforms, particularly in contrast to their pro-angiogenic counterparts, researchers can better understand the potential for developing targeted therapies that activate the natural inhibitory mechanisms of VEGF121b and VEGF165b.The exploration of these isoforms, therefore, opens new avenues for personalized medicine, where the specific angiogenic profile of a tumor could guide the choice of therapeutic intervention.Targeting VEGF isoforms such as VEGF-B, which can both suppress and facilitate angiogenesis depending on the physiological environment, represents a nuanced approach to anti-angiogenic therapy [36].
The family of VEGF splice variants is not random, but a carefully organized process influenced by various physiological and pathological stimuli.For example, hypoxia, a common feature of the tumor microenvironment, activates hypoxia-inducible factor-1 (HIF-1), which in turn can modulate VEGF splicing to favor the production of pro-angiogenic isoforms [37].In addition, splicing factors, including serine/arginine-rich proteins (SRs) and heterogeneous nuclear ribonucleoproteins (hnRNPs), bind VEGF mRNA and directly influence the splicing mechanism, thereby dictating the isoform outcome [38].The expression and dynamic activity of these splicing factors are fundamental in maintaining the delicate balance between pro-angiogenic and anti-angiogenic isoforms of VEGF, thus influencing angiogenic potential.

Molecular Perspective
At the molecular level, the interaction between VEGF splice variants and their respective receptors develops a complex network of intracellular signaling events.In the case of pro-angiogenic variants, binding to VEGFR2 is a critical step that triggers the activation of downstream signaling pathways involving multiple kinases and the induction of genes that control endothelial cell proliferation, migration, and survival [39].These processes are meticulously fine-tuned to ensure that angiogenesis is promoted in a controlled manner under normal physiological conditions.VEGFR1 has a higher binding affinity for VEGF-A isoforms, including both pro-angiogenic and anti-angiogenic variants, than VEGFR2.However, its role in angiogenesis is complex and multifaceted.One of the main functions of VEGFR1 is to act as a decoy receptor.By binding to high-affinity VEGF ligands, VEGFR1 can retain these molecules, preventing them from interacting with VEGFR2, which is the main driver of angiogenic signaling.This decoy function of VEGFR1 serves as a regulatory mechanism that modulates the intensity and duration of VEGFR2-mediated angiogenesis.For example, under conditions where excessive angiogenesis would be detrimental, such as in certain cancers, VEGFR1 may limit the availability of VEGF to VEGFR2, thereby reducing angiogenic signaling [39].
In addition, VEGFR1 may also function as a modulator of VEGFR2 activity.Although VEGFR1 itself has a weaker tyrosine kinase activity compared to VEGFR2, it may influence VEGFR2 signaling through ligand competition and receptor dimerization.When VEGF165b or other anti-angiogenic isoforms bind to VEGFR1, this may alter the receptor's interaction with VEGFR2, either by promoting heterodimer formation (VEGFR1/VEGFR2) or by modulating the availability of VEGFR2 to its pro-angiogenic ligands.This modulation may lead to altered downstream signaling, affecting processes such as endothelial cell proliferation, migration, and survival, which are essential for angiogenesis [40].
In the context of anti-angiogenic therapies, understanding the interplay between VEGFR1 and VEGFR2 is essential.Isoforms such as VEGF165b, which can bind to VEGFR1 without activating angiogenic pathways, may harness this decoy function to more effectively suppress VEGFR2-mediated signaling.This interaction emphasizes the therapeutic potential of targeting VEGFR1 together with VEGFR2 to achieve more precise modulation of angiogenesis in diseases such as cancer [39].In cancer, however, this balance is disturbed.HNSCC, like many other solid tumors, takes on the angiogenic capabilities of VEGF to enhance its own vascularization, which is essential for tumor growth and metastasis.Pro-angiogenic variants of VEGF are often upregulated in tumor cells, contributing to "angiogenic switching", a phenomenon in which the normal vasculature is transformed into a dynamic and disorganized network of blood vessels [41].
Anti-angiogenic splice variants, such as VEGF165b, provide a natural balance to this process.By binding to VEGF receptors with an affinity similar to that of pro-angiogenic variants, but without activating angiogenic signaling cascades, VEGF165b exerts an inhibitory effect.It effectively acts as a molecular trap, sequestering receptors and preventing pro-angiogenic variants from inducing their effects on endothelial cells.This unique mechanism highlights the potential of VEGF165b as a therapeutic agent, providing a way to undermine the aberrant angiogenesis seen in HNSCC and other cancers [42].
Understanding the splice variant profile of VEGF in tumors is important not only for the development of targeted therapies but also for prognostication and treatment planning.While current anti-angiogenic therapies focus primarily on inhibiting the activity of proangiogenic VEGF variants, resistance to these treatments is not uncommon.This is in part due to the ability of the tumor to adapt by finding alternative pathways to promote angiogenesis or by modulating the expression of VEGF variants [43].More effective therapeutic strategies can be developed by obtaining a deeper insight into the splicing mechanisms and regulatory factors that determine the VEGF isoform landscape within a tumor.
Future therapeutic approaches may include novel technologies such as gene editing or splice-switching oligonucleotides that can selectively alter VEGF mRNA splicing, thereby increasing the expression of anti-angiogenic variants such as VEGF165b [44,45].Such precision medicine strategies could provide more personalized and effective treatment options for patients with HNSCC, potentially improving outcomes while minimizing side effects.
The multifaceted interaction between different splice forms of VEGF represents a complex but promising frontier in cancer biology.The ability to modulate this balance by enhancing natural inhibitors of angiogenesis while reducing pro-angiogenic factors opens the door to innovative treatments for HNSCC.Further research into the regulation, function, and therapeutic targeting of VEGF splice variants will undoubtedly continue to shed light on the pathophysiology of angiogenesis and offer new hope to patients struggling with HNSCC.

Experimental Evidence of VEGF Splice Variants in Angiogenesis Inhibition
The study of VEGF splice variants has become increasingly important in understanding angiogenesis inhibition, particularly in the context of preclinical studies.Several investigators have explored the role of VEGF-A splicing mechanisms and their implications for anti-angiogenic therapeutics.For example, they have highlighted the critical nature of VEGF-A splicing in the development of anti-angiogenic therapies, suggesting that manipulation of these splicing events may provide novel therapeutic avenues [46].Furthermore, the discovery of alternatively spliced VEGFR-2 by Albuquerque et al. (2009) as a major endogenous inhibitor of lymphatic vessel growth highlights the complexity and importance of VEGF splicing variants in regulating angiogenesis.This receptor variant acts as a critical modulator and provides a potential target for therapeutic intervention in diseases characterized by abnormal lymphangiogenesis [47].
In addition, the interaction between VEGF and other molecular pathways has been the subject of intense research.For example, the VEGF-A/SOX2/SRSF2 network, as studied by Cherine Abou Faycal et al. ( 2019), plays a key role in the regulation of alternative splicing of VEGFR1 pre-mRNA in lung cancer cells, illustrating the complex regulatory mechanisms that control angiogenesis in cancer [48].Furthermore, Moens et al. (2014) discussed the multifaceted activity of VEGF in angiogenesis and its implications for therapeutic responses, highlighting the complexity of VEGF's role in promoting angiogenesis and how this may affect the efficacy of therapeutic strategies [49].
The study of VEGF splicing variants in angiogenesis inhibition is important not only for understanding the basic mechanisms of angiogenesis but also for developing targeted therapies that can effectively modulate these pathways to treat a variety of diseases, including cancer and vascular disorders.

Preclinical Studies
Over the past decade, preclinical studies have extensively explored the potential of VEGF165 as a therapeutic agent in the treatment of HNSCC.An important study by Zhang et al. ( 2018) introduced a VEGF165b variant with improved half-life and superior antitumor potency in a mouse model, demonstrating the potential for improved therapeutic interventions in cancer treatment.This variant exhibited extended stability and enhanced efficacy in inhibiting tumor angiogenesis, highlighting the value of targeting VEGF pathways in cancer therapy [50].
The interaction between VEGF165 and other molecular pathways has also been of interest.For example, Kim et al. (2017) explored how VEGFA links self-renewal and metastasis through the induction of Sox2 to repress miR-452 and Slug stimulation, providing insight into mechanisms to exploit VEGF165 therapeutically [51].Additionally, research efforts, such as those by Koyama et al. (2017), have attempted to normalize tumor vasculature by targeting VEGF pathways with the goal of restoring chemotherapeutic sensitivity in cancer models.This approach highlights the therapeutic potential of modulating VEGF165 expression and activity to improve treatment outcomes [52].
These and other studies in the field continue to provide valuable insights into the role of VEGF165 in cancer biology and its potential as a target for therapeutic intervention.By understanding the mechanisms by which VEGF165 affects angiogenesis and tumor growth, researchers are developing more effective strategies to combat HNSCC and other cancers.

Clinical Perspectives on VEGF165 as a Treatment for HNSCC
In recent years, clinical research on VEGF165 has gained significant attention, particularly in its application as a therapeutic target in HNSCC.VEGF165 has been the focus of various studies aimed at inhibiting tumor growth and metastasis by modulating blood vessel formation.A notable study by Vassilakopoulou et al. (2015) focused on targeting angiogenesis in HNSCC, emphasizing the potential of VEGF165 inhibitors to improve treatment outcomes for patients with this aggressive cancer.This research underscores the importance of angiogenesis in cancer progression and the therapeutic benefits of inhibiting the VEGF pathway [53].In the field of differentiated thyroid carcinoma (DTC), Abdel Rahman (2015) explored the efficacy of targeting the VEGF pathway in iodine-refractory DTC, demonstrating the potential of VEGF165 inhibitors to provide a therapeutic advantage in cases where conventional treatments are insufficient [54].
Neufeld et al.'s (1999) comprehensive review of VEGF and its receptors further elucidates the mechanistic basis of VEGF-mediated angiogenesis, providing insight into how clinical interventions targeting VEGF165 could be optimized to enhance antitumor effects while minimizing adverse outcomes [55].In addition, Ashina et al. (2015) provided evidence that VEGF-induced increases in blood flow cause vascular hyperpermeability in vivo, contributing to our understanding of how modulation of VEGF165 may impact the tumor microenvironment and treatment efficacy [56].
These clinical studies highlight the therapeutic potential of targeting VEGF165 in the treatment of cancer and provide new avenues for the development of effective antiangiogenic therapies.By inhibiting VEGF-mediated pathways, researchers aim to deprive tumors of blood supply and thereby inhibit growth and metastasis.As our understanding of the role of VEGF165 in cancer biology deepens, new therapeutic strategies are expected to emerge and improve outcomes for patients with HNSCC and other cancers.

Potential Limitations and Future Directions
Therapeutic targeting of VEGF splice variants for the treatment of cancer and other diseases represents a promising avenue for medical research and patient care.However, this approach is not without limitations and challenges (Figure 2).The complexity of VEGF signaling and its indispensable role in physiological and pathological angiogenesis requires a nuanced understanding of the potential obstacles to the development of effective therapies [33].
pathway in iodine-refractory DTC, demonstrating the potential of VEGF165 inhibitors to provide a therapeutic advantage in cases where conventional treatments are insufficient [54].Neufeld et al.'s (1999) comprehensive review of VEGF and its receptors further elucidates the mechanistic basis of VEGF-mediated angiogenesis, providing insight into how clinical interventions targeting VEGF165 could be optimized to enhance antitumor effects while minimizing adverse outcomes [55].In addition, Ashina et al. (2015) provided evidence that VEGF-induced increases in blood flow cause vascular hyperpermeability in vivo, contributing to our understanding of how modulation of VEGF165 may impact the tumor microenvironment and treatment efficacy [56].
These clinical studies highlight the therapeutic potential of targeting VEGF165 in the treatment of cancer and provide new avenues for the development of effective antiangiogenic therapies.By inhibiting VEGF-mediated pathways, researchers aim to deprive tumors of blood supply and thereby inhibit growth and metastasis.As our understanding of the role of VEGF165 in cancer biology deepens, new therapeutic strategies are expected to emerge and improve outcomes for patients with HNSCC and other cancers.

Potential Limitations and Future Directions
Therapeutic targeting of VEGF splice variants for the treatment of cancer and other diseases represents a promising avenue for medical research and patient care.However, this approach is not without limitations and challenges (Figure 2).The complexity of VEGF signaling and its indispensable role in physiological and pathological angiogenesis requires a nuanced understanding of the potential obstacles to the development of effective therapies [33].One of the major challenges in targeting VEGF splice variants is the specificity of therapeutic agents.VEGF and its variants are involved in both healthy and pathologic states, contributing to wound healing, normal vascular function, and pathologic angiogenesis [57].Developing therapies that selectively inhibit the pathological functions of VEGF splice variants without interfering with their physiological roles remains a significant challenge.As with many targeted cancer therapies, there is a potential for resistance to treatments targeting VEGF splice variants.Tumors may be able to activate alternative angiogenic pathways or mechanisms to circumvent the inhibition of VEGF signaling, and thus reduce the efficacy of these targeted therapies over time [58,59].
Targeting VEGF splice variants may result in adverse effects due to the inhibition of normal angiogenesis and vascular maintenance.Potential side effects include impaired wound healing, hypertension, proteinuria, and increased risk of thromboembolic events.These risks require careful consideration and management in the clinical setting [60].The heterogeneity of tumors is another challenge.Expression levels and roles of VEGF splice variants can vary significantly between cancers and within tumors of the same type.This variability may influence the responsiveness of tumors to VEGF-targeted therapies and complicate the development of generally effective treatments [61].
Efficient delivery of therapeutic agents targeting VEGF splice variants to the tumor site is critical to their success.However, abnormal and often inefficient tumor vasculature can impede the path and distribution of these agents, limiting their therapeutic potential [62].The development of therapies targeting VEGF splice variants also faces regulatory and ethical considerations.Rigorous clinical trials are required to demonstrate the safety and efficacy of these treatments and the high costs associated with research and development may impact the accessibility and affordability of approved therapies [63].
Despite these challenges, the limitations associated with targeting VEGF splice variants continue to be addressed through continued research and technological advances.To overcome these obstacles and improve the efficacy of treatments for cancer and other angiogenesis-related diseases, strategies such as combination therapies, advanced drug delivery systems, and personalized medicine approaches are being explored.

Pro-Angiogenic vs. Anti-Angiogenic VEGF Variants in HNSCC: Clinical Impact
VEGF has long been recognized as an essential regulator of angiogenesis and plays a key role in carcinoma progression.This complex role is further complicated by the existence of VEGF splice variants that can promote (pro-angiogenic) or inhibit (anti-angiogenic) angiogenesis, thereby influencing the tumor microenvironment and response to therapy.While pro-angiogenic variants, such as VEGF-A, are well studied for their role in facilitating tumor growth and metastasis by promoting the formation of new blood vessels, antiangiogenic splice variants, represented by VEGF165b, provide a natural countermeasure by inhibiting these processes [39].This section aims to outline the clinical implications of these opposing forces in the context of HNSCC, setting the stage for a discussion of how utilizing the balance between pro-angiogenic and anti-angiogenic VEGF variants may provide new avenues for targeted therapies.The intricate interplay between these variants not only underscores the complexity of tumor angiogenesis but also demonstrates the potential for personalized treatment strategies that could improve patient outcomes in HNSCC.
In the field of HNSCC, the dichotomy between pro-and anti-angiogenic VEGF variants is emerging as an essential aspect of tumor biology and therapeutic targeting.In particular, the VEGF165 variant exemplifies the pro-angiogenic group, which promotes tumor growth and metastasis by increasing angiogenesis.This variant, by binding to VEGFR2, triggers an intracellular signaling cascade that culminates in endothelial cell proliferation, migration, and new blood vessel formation, thereby facilitating tumor progression [64].
In contrast, VEGF165b, a splice variant of VEGF-A, is unique among anti-angiogenic factors in its ability to bind to VEGF receptors without activation of downstream proangiogenic pathways.This interaction effectively blocks angiogenic signals that would otherwise be mediated by pro-angiogenic variants, illustrating a natural mechanism of angiogenesis inhibition in the tumor microenvironment [65].The implications of these opposing functions are profound, affecting not only cancer progression but also the efficacy and development of anti-angiogenic therapies.
Recent studies have revealed the complex role these variants play in cancer.For example, research has shown that altering the balance between pro-and anti-angiogenic VEGF variants can have a significant impact on disease progression and response to treatment.Therapies that shift this balance toward anti-angiogenesis have shown promise in preclinical models of HNSCC, suggesting a potential pathway for the development of novel treatment strategies that exploit the inherent anti-angiogenic properties of variants such as VEGF165b [66].
In this complicated context of tumor angiogenesis, particularly in HNSCC, the distinction between pro-angiogenic and anti-angiogenic VEGF variants provides crucial insight into therapeutic targeting and disease progression.Table 2 below consolidates these variants, delineating their distinct roles, clinical relevance, and basis for further investigation, thus providing a foundation for understanding the potential for targeted modulation of angiogenesis in HNSCC therapy.
Table 2. Comparative analysis of VEGF variants in angiogenesis, detailing their pro-angiogenic and anti-angiogenic actions.
Associated with tumor progression and metastasis in various cancers, including HNSCC.

VEGF-A121
[68] Pro-angiogenic Similar to VEGF-A165 but more diffusible due to the lack of heparin-binding domains.
Plays a role in angiogenesis and tumor growth.
Influences the angiogenic profile in specific tissue environments.
Inhibits angiogenesis, offering a potential therapeutic target for reducing tumor growth and angiogenesis in cancers.
Potentially reduces angiogenesis and tumor progression, similar to VEGF-A165b.

VEGF-B [35]
Pro-angiogenic Binds primarily to VEGFR1, involved in heart development and fatty acid uptake.
Role in cancer is less clear but may be involved in metabolic regulation and survival of cancer cells.
Implicated in lymphatic metastasis of solid tumors, including HNSCC, by promoting lymphangiogenesis.

VEGF-D [72]
Pro-angiogenic Similar to VEGF-C, promotes lymphangiogenesis and angiogenesis by binding to VEGFR2 and VEGFR3.
Potential role in lymphatic spread and metastasis of cancer, including implications for HNSCC.
Studied for its potential in cancer therapy and cardiovascular diseases, though with varying implications in different types of cancer.
The balance between pro-and anti-angiogenic variants of VEGF plays an important role in the angiogenic landscape of head and neck squamous cell carcinoma.Pro-angiogenic variants, particularly VEGF-A, facilitate tumor progression by promoting vascular permeability and endothelial cell proliferation, leading to the formation of new blood vessels [58].In contrast, anti-angiogenic variants, such as VEGF165b, act as natural inhibitors of angiogenesis, potentially limiting tumor growth and spread by binding to VEGF receptors without activating downstream pro-angiogenic pathways [74].
Therapeutic manipulation of this balance to suppress tumor angiogenesis while promoting the body's natural anti-angiogenic mechanisms is a promising approach to the treatment of HNSCC.The targeting of VEGF-specific signaling pathways with monoclonal antibodies or tyrosine kinase inhibitors has been shown to be effective in reducing vascularization and tumor growth.However, developing resistance to these agents requires a deeper understanding of the complex interplay between VEGF variants and the tumor microenvironment [75].
The clinical implications of the pro-and anti-angiogenic balance of VEGF variants are profound.The efficacy of existing therapies, in particular anti-angiogenic drugs, can be significantly influenced by this balance, with an imbalance in favor of the pro-angiogenic variants being associated with aggressive tumor growth and a poorer outcome in patients.Resistance to anti-angiogenic therapy, which is a major challenge in the treatment of HNSCC, is often due to tumor adaptation strategies, such as the increase in the number of alternative angiogenic factors or receptors, which render the treatments ineffective over time [50].
Personalizing therapeutic approaches based on each patient's VEGF variant profile could improve treatment efficacy and overcome resistance.Biomarker-based strategies, including quantifying pro-and anti-angiogenic VEGF variants in tumor samples or circulating blood, offer a potential method for more effectively personalizing therapy.Such personalized interventions could improve patient response and minimize side effects by avoiding unnecessary broad-spectrum anti-angiogenic exposure [76].
Moving forward, the development of next-generation therapies for HNSCC will depend on advances in understanding the biology of VEGF and the mechanisms underlying resistance to anti-angiogenic therapy.Elucidating the precise roles of different VEGF variants in tumor angiogenesis and exploring novel therapeutic targets within these pathways should be the focus of future research.Additionally, the implementation of advanced diagnostic techniques to assess VEGF variant profiles promises to refine treatment strategies, paving the way for more effective and durable HNSCC responses.

Resistance Mechanisms and Overcoming Therapeutic Challenges
A major advance in the field of oncology has been the development of anti-angiogenic therapies targeting VEGF splice variants in the context of cancer treatment.However, the emergence of resistance to these therapies represents a significant challenge, requiring a deeper understanding of the underlying mechanisms and the exploration of strategies to overcome these barriers, such as a detailed examination of how tumors can avoid VEGF blockade by activating other angiogenic pathways, such as those mediated by fibroblast growth factors (FGFs) or angiopoietins.This compensatory mechanism undermines the efficacy of VEGF-targeted treatments and is a major obstacle in cancer therapy [77].
It is important to consider vascular co-option, which is a process by which tumors make use of existing blood vessels rather than forming new ones.This mechanism of resistance to anti-angiogenic therapy is particularly prevalent in liver metastases and presents a challenge to the efficacy of VEGF-targeted approaches [78].The tumor microenvironment also has an important role in resistance.Further complicating the therapeutic landscape, tumor hypoxia can induce the expression of other pro-angiogenic factors.Immune cells in the tumor microenvironment can create immunosuppressive environments supporting tumor growth and angiogenesis [79].A multifaceted approach is needed to overcome these challenges.It requires not only understanding the underlying mechanisms of resistance but also developing strategies to counter these mechanisms.This could include combination therapies that target multiple angiogenic pathways, modulate the tumor microenvironment, or enhance the immune response against tumor cells.
Research has shown that the diversity of VEGF splice variants provides new insights for targeted therapy.Montemagno et al. (2023) demonstrated that novel VEGF splice variants in renal cell carcinoma are less effectively inhibited by conventional anti-VEGF/VEGFR therapies, suggesting that these variants may serve as alternative therapeutic targets for patients resistant to current treatments.Their study highlights the importance of understanding the variability in the expression of VEGF splice variants and how it affects patient-specific treatment strategies [59].
Subsequent breast cancer research has shown that VEGF-A and its splice variants significantly influence tumor development, with clinical implications that could potentially refine therapeutic approaches.According to Kawas et al. (2022), the roles of these splice variants provide a deeper understanding of their involvement in breast cancer progression and a basis for the development of splice variant-specific therapies.This approach could enhance the efficacy of anti-angiogenic treatments and improve patient outcomes [80].
A further level of complexity in the approach to angiogenesis is introduced by the different binding affinities of VEGF-A splice variants to VEGF receptors (VEGFR).Mamer et al. (2020) showed that these variants bind VEGFR with different affinities and thus differentially affect angiogenic signaling pathways.This finding paves the way for designing more precise anti-angiogenic agents that selectively inhibit specific VEGF/VEGFR interactions, potentially reducing effects and increasing therapeutic efficacy [81].
The appearance of VEGF165b, a splice variant of VEGF-A, was particularly remarkable.Boudria et al. (2018) demonstrated its role in the promotion of lung tumor progression and resistance to anti-angiogenic therapies.VEGF165b functions through an autocrine β1 integrin/VEGFR feedback loop, increasing tumor aggressiveness and circumventing the effects of current anti-angiogenic drugs.Targeting this splice variant may provide a novel strategy to overcome resistance and achieve better control of tumor progression [33].Prince et al. (2019) shed light on novel approaches to inhibit VEGF165-mediated angiogenic pathways, offering hope for more effective treatments for HNSCC.For example, adjuvant anti-angiogenic therapy targeting VEGFR2 and VEGFR3 has shown promise in improving chemotherapeutic uptake in HNSCC models.The goal of this approach is to inhibit tumor vascularization and therefore improve the delivery and efficacy of chemotherapeutic agents [82].
Furthermore, the combination of anti-PD-1 monoclonal antibodies with anti-VEGF agents has emerged as a safe and effective strategy for the second-line or subsequent therapy of recurrent or metastatic HNSCC.This combination therapy not only directly targets tumor cells, but also disrupts the blood supply to the tumor.This provides a multifaceted attack against the cancer.The importance of VEGF and its splice variants in the pathogenesis and treatment of HNSCC is underscored by the study of angiogenesis and anti-angiogenic therapy in HNSCC [83].The understanding of the specific roles and mechanisms of action of VEGF165 in tumor angiogenesis will provide a solid foundation for the development of targeted therapies that could significantly improve the outcome of patients with HNSCC.
The outcome would underscore the importance of understanding the mechanisms of resistance to therapies targeting VEGF and the need to develop multifaceted strategies to overcome these challenges.This would underscore the continued need for research to identify new targets, understand the tumor microenvironment, and tailor treatments to individual patient profiles to improve outcomes in cancer therapy.

Conclusions
In conclusion, this review presents the importance of VEGF splice variants, in particular VEGF165b, in inhibiting angiogenesis in the context of head and neck squamous cell carcinoma.VEGF165b is a promising therapeutic target due to its unique anti-angiogenic properties and its divergence from the pro-angiogenic activities typically attributed to VEGF isoforms.Further exploration of VEGF splice variants as tools for personalized medicine is essential and may reshape the management of HNSCC.This review also recognizes the challenges facing the VEGF splice variant approach, including target specificity, resistance to therapy, adverse effects, cancer heterogeneity, and regulatory and ethical considerations.

Figure 1 .
Figure 1.VEGF signaling pathways.VEGF binding to VEGFR activates several downstream signaling cascades, including the MAPK pathway (RAF-MEK-MAPK), leading to cell proliferation and survival, and the PI3K pathway (PI3K-AKT), promoting cell growth and survival.These pathways contribute to angiogenesis and are critical in the context of tumor progression.

Figure 1 .
Figure 1.VEGF signaling pathways.VEGF binding to VEGFR activates several downstream signaling cascades, including the MAPK pathway (RAF-MEK-MAPK), leading to cell proliferation and survival, and the PI3K pathway (PI3K-AKT), promoting cell growth and survival.These pathways contribute to angiogenesis and are critical in the context of tumor progression.

Figure 2 .
Figure 2. Focusing on the key issues of target specificity, each element represents a critical factor influencing the efficacy and outcome of cancer treatments.

Figure 2 .
Figure 2. Focusing on the key issues of target specificity, each element represents a critical factor influencing the efficacy and outcome of cancer treatments.

Table 1 .
The table indicates that while VEGF121, VEGF165, and VEGF189 are pro-angiogenic, promoting the formation of new blood vessels, VEGF165b is anti-angiogenic, inhibiting the angiogenic pathways.

Table 1 .
The table indicates that while VEGF121, VEGF165, and VEGF189 are pro-angiogenic, promoting the formation of new blood vessels, VEGF165b is anti-angiogenic, inhibiting the angiogenic pathways.