VEGF Human (121 a.a.), His

What is the molecular structure of VEGF-121 and how does it differ from other VEGF isoforms?

VEGF-121 is a diffusible protein that arises from alternative splicing of the VEGF gene. It lacks exons 6 and 7, making it structurally distinct from other VEGF isoforms. The protein is a homodimer consisting of two identical non-glycosylated polypeptide chains, each containing 121 amino acid residues without N-terminal methionine, with a total molecular mass of approximately 28.4 kDa .

Unlike other major VEGF isoforms (VEGF-165, VEGF-189, and VEGF-206), VEGF-121 lacks the heparin-binding domain, making it a freely diffusible protein. This characteristic impacts its spatial distribution and biological activity in tissues. VEGF-121 is a weakly acidic polypeptide since it lacks 15 basic amino acids within the 44 residues encoded by exon 7 .

Table 1: Comparison of Key VEGF Isoforms

How does VEGF-121 interact with its receptors, and what signaling pathways does it activate?

VEGF-121 primarily binds to two receptor tyrosine kinases: VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). Upon binding, it induces receptor dimerization and autophosphorylation of tyrosine 1173 (Y1175 in humans), which can be readily detected in intact endothelial cells . Phosphorylation of tyrosine residues 1054 and 1059 in the kinase domain are required for receptor activation, as they maintain the open configuration of the ATP-binding pocket of the tyrosine kinase .

Recent research has revealed more complex interactions with neuropilins (Np). While initially it was thought that VEGF-121 did not interact with Np-1, newer studies demonstrate that:

• Np-1 and Np-2 can enhance VEGF-121-stimulated signal transduction via phosphorylation of VEGFR-2

• Blocking functional Np-1 reduces VEGF-121-induced endothelial cell migration and sprout formation

• VEGF-121 does bind directly to Np-1, though this interaction alone is not sufficient to create the Np-1/VEGFR-2 complex

These interactions trigger downstream signaling cascades that promote endothelial cell proliferation, migration, and survival.

What are the optimal conditions for reconstitution and storage of recombinant VEGF-121?

Proper handling of recombinant VEGF-121 is crucial for maintaining its biological activity. Based on manufacturer guidelines, the following protocol is recommended:

Storage:

• Lyophilized VEGF-121 should be stored at -20°C

• The expiration date is typically indicated on the vial label

• Upon reconstitution, aliquots should be stored at -20°C or below

• Repeated freeze-thaw cycles should be avoided

Reconstitution:

• Reconstitute lyophilized VEGF-121 with deionized sterile-filtered water to a final concentration of 0.1–1.0 mg/mL

• Use a minimal initial volume of 100 μL

• For further dilutions, prepare with 0.1% bovine serum albumin (BSA) or human serum albumin (HSA) in phosphate-buffered saline

Quality control parameters indicate that properly prepared VEGF-121 should have:

• Purity >97% as determined by SDS-PAGE analysis

• Low endotoxin levels (<1 EU/μg cytokine) as determined by Limulus Amebocyte Lysate (LAL) assay

• Biological activity with ED50 ≤0.4 ng/mL, corresponding to an activity of ≥2.5×10^6 U/mg

How can VEGF-121 be used for imaging ischemic tissues in experimental models?

VEGF-121 offers a unique approach to identifying tissues experiencing hypoxic stress due to its specific binding to upregulated VEGF receptors in ischemic microvasculature. The methodology involves:

• Radiolabeling: VEGF-121 is labeled with radioisotopes such as Indium-111 (^111In)

• Administration: The labeled protein is administered intravenously (typical dose: 100 μCi of ^111In-labeled recombinant human VEGF-121)

• Imaging: Biodistribution studies and planar imaging are conducted at specific time points (e.g., 3, 24, and 48 hours post-injection)

• Analysis: Quantification of radioactivity in regions of interest and comparison with control tissues

In rabbit models of unilateral hindlimb ischemia, this approach demonstrated:

• Greater accumulation of ^111In-labeled VEGF-121 in ischemic compared to control tissue (p<0.02)

• Significantly higher radioactivity in ischemic muscle compared to sham-operated and contralateral non-operated hindlimbs at 3 hours post-injection (p<0.02)

• Specificity of targeting confirmed by the absence of differential uptake when using ^125I-labeled human serum albumin as a control

Immunohistochemical staining confirmed that this targeting corresponds to upregulation of VEGF receptors in ischemic skeletal muscle. This approach has significant potential for monitoring the efficacy of revascularization strategies, including therapeutic angiogenesis .

How does VEGF-121 expression change in tumor angiogenesis and cancer progression?

VEGF-121 plays a significant role in tumor angiogenesis with distinct expression patterns across different cancer types:

In prostate cancer:

• Normal prostate tissue shows a balance of isoform expression favoring VEGF-165 over VEGF-121

• Malignant prostate tissue exhibits a significant shift toward VEGF-121 expression

• Increased relative amounts of VEGF-121 correlate with enhanced prostate tumor angiogenesis

In colon cancer:

• VEGF-121 is upregulated and hypothesized to play an important role in the proliferative, angiogenic process due to its high bioavailability

• Other splice variants (VEGF-165, VEGF-189, and VEGF-145) are also consistently expressed

In non-small-cell lung carcinoma:

• VEGF-121 promotes lymphangiogenesis in the sentinel lymph nodes

• It may act, at least in part, via the induction of VEGF-C

In glioblastoma:

These findings suggest that VEGF-121 could serve as both a biomarker and potential therapeutic target in various cancers.

What is the role of VEGF-121 in physiological and pathological angiogenesis?

VEGF-121 contributes to angiogenesis through several mechanisms:

• Promotion of endothelial cell proliferation

• Enhancement of macromolecular extravasation

• Stimulation of vascular permeability

• Induction of endothelial cell migration and sprout formation

The spatial distribution of VEGF isoforms, affected by their differing heparin-binding affinities, determines whether blood vessel growth is organized and directed, or disordered. Studies in mice engineered to express only VEGF-120 (mouse equivalent of human VEGF-121) showed:

• Significant decrease in capillary branch formation

• Disruption of blood vessel growth

• Impairment of directed extension of endothelial cell filopodia during neural tube development

Similarly, in mice lacking VEGF-164 and VEGF-188 isoforms (relying primarily on VEGF-120), severe defects in retinal vascular outgrowth were observed .

In non-cancer pathologies, VEGF-121 plays a role in preeclampsia, where it has been shown to attenuate hypertension and improve kidney damage in rat models of the condition .

How can VEGF-121 plasma levels be used as biomarkers for response to anti-angiogenic therapy?

The utilization of VEGF-121 plasma levels as biomarkers for anti-angiogenic therapy response represents an emerging area of research:

In brain xenograft models of human glioblastoma cells:

• Plasma VEGF-121 levels correlate with tumor volume

• Intravenous infusion of bevacizumab significantly decreases plasma VEGF-121 levels

In human patients with recurrent glioblastoma:

Table 2: Correlation Between VEGF-121 Plasma Levels and Clinical Outcomes

The mechanism proposed for this correlation suggests that circulating VEGF-121 may reduce the amount of bevacizumab available to target the heavier isoforms of VEGF, which are considered more clinically relevant in the tumor microenvironment .

What experimental models are most suitable for studying VEGF-121 function in research?

Various experimental models have proven valuable for investigating VEGF-121 function:

In Vitro Models:

• Human Umbilical Vein Endothelial Cells (HUVECs):

  • Used for determining biological activity (ED50)

  • Valuable for studying dose-dependent stimulation effects

  • Essential for investigating endothelial cell migration and tube formation

• Receptor binding assays:

  • Analysis of interactions with VEGFR-1, VEGFR-2, and neuropilins

  • Investigation of signal transduction pathways

  • Study of receptor dimerization and phosphorylation events

In Vivo Models:

• Rabbit model of unilateral hindlimb ischemia:

  • Created by femoral artery excision

  • Effective for studying VEGF-121 targeting to ischemic tissue

  • Valuable for developing imaging approaches for ischemia detection

• Brain xenograft models:

  • Using human glioblastoma cell lines (e.g., U87MG)

  • Allows correlation between tumor volume and plasma VEGF-121 levels

  • Useful for evaluating anti-angiogenic therapy responses

• Genetically modified mice:

  • Mice expressing only specific VEGF isoforms

  • Study of distinct roles in vascular development

  • Investigation of compensatory mechanisms in the absence of specific isoforms

These models provide complementary information and can be selected based on specific research questions regarding VEGF-121 function, regulation, or therapeutic applications.

What are the emerging therapeutic applications of VEGF-121 in ischemic diseases?

The unique properties of VEGF-121, particularly its high diffusibility and specific binding to upregulated receptors in ischemic tissues, make it a promising candidate for several therapeutic applications:

• Diagnostic imaging of ischemic tissues:

  • Radiolabeled VEGF-121 can identify ischemic but viable tissue in atherosclerotic cardiovascular disease

  • This approach could help in selecting appropriate revascularization therapies and assessing their efficacy

• Therapeutic angiogenesis:

  • Delivery of VEGF-121 to promote revascularization in ischemic tissues

  • Potential applications in peripheral arterial disease, coronary artery disease, and stroke

  • The freely diffusible nature of VEGF-121 could provide advantages for distribution throughout ischemic regions

• Combined diagnostic and therapeutic strategies:

  • Using VEGF-121 as both a targeting moiety and a therapeutic agent

  • Development of theranostic approaches that simultaneously diagnose and treat ischemic conditions

Future research should focus on optimizing delivery methods, determining ideal dosing regimens, and evaluating the long-term safety and efficacy of VEGF-121-based interventions in various ischemic conditions.

How might the molecular diversity of VEGF isoforms, including VEGF-121, be harnessed for personalized medicine approaches?

The molecular diversity of VEGF isoforms presents opportunities for developing personalized medicine approaches:

• Isoform profiling as prognostic biomarkers:

  • Analysis of the VEGF isoform expression pattern in tumors or plasma could predict disease progression

  • Higher VEGF-121 levels in glioblastoma patients correlate with poorer outcomes, suggesting its potential as a stratification marker

• Predictive biomarkers for therapy selection:

  • VEGF isoform profiles might predict response to anti-angiogenic therapies

  • Patients with elevated VEGF-121 might benefit from higher doses of bevacizumab or alternative therapeutic approaches

• Targeted therapeutic development:

  • Design of agents specifically targeting particular VEGF isoforms

  • Development of isoform-switching modulators to restore normal VEGF isoform balance

• Combination therapies:

  • Targeting multiple VEGF isoforms simultaneously

  • Combining VEGF isoform-specific therapies with other treatment modalities

As our understanding of the distinct roles of each VEGF isoform expands, so does the potential for more precise and effective therapeutic interventions tailored to individual patients' molecular profiles.