VEGF Rat, Yeast

What is the baseline expression pattern of VEGF in healthy rat tissues?

VEGF is expressed in various rat tissues under normal conditions, with particularly notable expression in islets of Langerhans. Research demonstrates that freshly isolated rat islets express VEGF at baseline levels that can be quantified using in situ hybridization techniques. Moreover, isolated rat islets express specific VEGF isoforms, with VEGF120 and VEGF164 being the predominant variants detected through reverse transcriptase-polymerase chain reaction (RT-PCR) . Tissue-specific distribution patterns of VEGF isoforms can be observed across different rat organs, with differential expression of VEGF-Axxxa and VEGF-Axxxb splice variants depending on the tissue type .

How does tissue hypoxia affect VEGF expression in rat models?

Hypoxic conditions significantly upregulate VEGF expression in rat tissues. When islet tissue is exposed to hypoxic/anoxic conditions for 8 hours, Northern blot hybridization analysis reveals a substantial 3.7-fold increase in VEGF mRNA expression compared to normoxic controls . This hypoxia-induced upregulation represents an important physiological response that may facilitate angiogenesis and revascularization in oxygen-deprived tissues. Interestingly, while primary islet tissue shows hypoxia-responsive VEGF expression, certain rodent insulinoma cell lines (RINm5F-2A, INS-1, and MIN6) that express VEGF do not demonstrate modulated expression under hypoxic/anoxic conditions .

Does VEGF expression in rat tissues change over time in culture?

Yes, isolated rat islet tissue demonstrates dynamic changes in VEGF expression during in vitro culture. When analyzed by in situ hybridization, islet tissue exhibits a significant 4.6-fold increase in VEGF mRNA expression over time in culture from day 0 to day 7 . This temporal upregulation suggests that devascularization during the isolation process triggers compensatory VEGF expression, potentially as a mechanism to promote revascularization. This observation has important implications for transplantation studies, as it indicates that manipulating pre-transplant conditions could potentially enhance VEGF expression and improve islet survival.

What are the primary VEGF splice variants expressed in rat models?

Rat tissues express multiple VEGF splice variants with distinct molecular characteristics. Reverse transcriptase-polymerase chain reaction analysis reveals that freshly isolated rat islets primarily express two VEGF isoforms: VEGF120 and VEGF164 . In lower urinary tract tissues, research has identified both VEGF-Axxxa and VEGF-Axxxb splice variant families, though VEGF-Axxxa predominates at the mRNA level in all examined lower urinary tract tissues . This differential expression pattern suggests tissue-specific regulation of alternative splicing mechanisms governing VEGF isoform expression.

How do expression patterns of VEGF splice variants change during inflammatory conditions?

During inflammatory conditions such as cyclophosphamide (CYP)-induced cystitis, both VEGF-Axxxa and VEGF-Axxxb splice variants show altered expression patterns in rat lower urinary tract tissues. Studies demonstrate that both isoform families increase in lower urinary tract tissues following CYP-induced cystitis, with tissue-specific patterns of upregulation . This inflammation-induced change in VEGF isoform balance may contribute to pathological changes in tissue function, potentially through altered angiogenic signaling or direct effects on neural sensitization.

What methodological approaches can accurately quantify different VEGF splice variants in rat tissues?

Several complementary methodological approaches can be employed to accurately quantify VEGF splice variants in rat tissues:

• Quantitative RT-PCR: This technique allows precise quantification of mRNA expression of specific VEGF isoforms. Using primers designed to amplify unique regions of each splice variant, researchers can determine relative abundance of each isoform. For standardized presentation, expression values can be normalized to housekeeping genes (L32 or 18S) and presented relative to control conditions .

• Northern Blot Hybridization: This technique provides visualization and quantification of specific VEGF mRNA transcripts. It has been successfully used to demonstrate the 3.7-fold increase in VEGF mRNA under hypoxic conditions in rat islet tissue .

• In Situ Hybridization: This approach allows visualization of VEGF mRNA expression in intact tissue sections, enabling spatial localization of expression patterns .

• Western Blot Analysis: Using isoform-specific antibodies, Western blotting can detect and quantify VEGF protein isoforms, as demonstrated with the ability to detect recombinant rat VEGF164 in experimental samples .

What VEGF receptors are expressed in rat islet tissue?

Rat islet tissues express specific VEGF tyrosine kinase receptors that mediate VEGF signaling. Reverse transcriptase-polymerase chain reaction analysis has revealed the presence of both flt (VEGFR1) and flk-1 (VEGFR2) receptors in freshly isolated rat islets . This co-expression of multiple VEGF receptor subtypes suggests complex regulation of VEGF-mediated effects in islet tissue, potentially involving different downstream signaling pathways and biological responses depending on which receptor is predominantly engaged.

How does blocking VEGFR2 signaling affect bladder function in rat models?

Blockade of VEGFR2 signaling produces significant functional effects on bladder physiology in rat models. When Ki-8751, a potent and selective VEGFR2 tyrosine kinase inhibitor, is administered intravesically at 1 mg/kg, multiple urodynamic parameters are altered:

• Bladder Capacity: VEGFR2 blockade significantly increases bladder capacity in both male and female rats. Specifically:

  • In control (no CYP) rats: 1.3-fold increase in females (p≤0.01) and 1.4-fold increase in males (p≤0.01)

  • In acute CYP-induced cystitis: 1.4-fold increase in females (p≤0.05) and 1.5-fold increase in males (p≤0.05)

  • In chronic CYP-induced cystitis: 1.6-fold increase in females (p≤0.05) and 2.1-fold increase in males (p≤0.01)

• Void Volume: VEGFR2 antagonism increases void volume in multiple experimental groups:

  • In control rats: 1.2-fold increase in females (p≤0.01) and 1.3-fold increase in males (p≤0.05)

  • In acute CYP-induced cystitis: 1.3-fold increase in females (p≤0.05) but no significant change in males

  • In chronic CYP-induced cystitis: 1.4-fold increase in females (p≤0.05) and 2.2-fold increase in males (p≤0.01)

These findings suggest that endogenous VEGF/VEGFR2 signaling plays a significant role in regulating normal bladder function and contributes to bladder dysfunction during inflammatory conditions.

Are there sex differences in VEGF/VEGFR2 signaling responses in rat models?

Yes, research indicates sex-specific differences in the response to VEGF/VEGFR2 signaling blockade in rat models. While both male and female rats show functional changes after VEGFR2 antagonism, the magnitude of these changes differs between sexes, particularly in inflammatory conditions. In female rats, the effect of VEGFR2 blockade is relatively consistent across control and CYP-induced cystitis conditions. In contrast, male rats show a graduated response, with the greatest increases in bladder capacity and void volumes observed in chronic cystitis, followed by acute cystitis, and finally control conditions . This suggests that VEGF/VEGFR2 signaling may have sex-specific roles in bladder function regulation, particularly during inflammatory conditions.

What are the optimal techniques for detecting VEGF protein in rat tissue samples?

Several techniques can be employed for optimal detection of VEGF protein in rat tissue samples:

• Western Blot Analysis: Using specific anti-rat VEGF antibodies, Western blotting can detect VEGF protein isoforms in tissue lysates. Polyclonal antibodies such as Goat Anti-Rat VEGF164 Antigen Affinity-purified Polyclonal Antibody have been validated for detecting recombinant rat VEGF164 in Western blot applications .

• Immunohistochemistry (IHC): For spatial localization of VEGF protein in tissue sections, IHC using specific antibodies can be performed. For example, VEGF164 has been successfully detected in perfusion-fixed frozen sections of rat kidney using Goat Anti-Rat VEGF164 Antigen Affinity-purified Polyclonal Antibody (15 μg/mL) with appropriate visualization systems such as HRP-DAB staining .

• Enzyme-Linked Immunosorbent Assay (ELISA): While not specifically mentioned in the search results, ELISA represents another quantitative approach for measuring VEGF protein levels in tissue homogenates or biological fluids.

• Functional Bioassays: VEGF bioactivity can be assessed using functional assays, such as the endothelial cell invasion and capillary morphogenesis assay that has been used to demonstrate bioactivity of VEGF derived from RINm5F-2A cells .

How can researchers quantify VEGF mRNA expression in rat tissues?

Several molecular techniques can be employed to quantify VEGF mRNA expression in rat tissues:

• Quantitative Real-Time PCR (qRT-PCR): This technique allows precise quantification of VEGF mRNA expression. The methodology involves:

  • RNA extraction from tissues

  • cDNA synthesis using reverse transcriptase

  • Amplification using SYBR Green I chemistry with specific primers

  • Thermal cycling protocol: 94°C for 2 min, followed by 45 cycles at 94°C for 15 s and 56-60°C for 30 s

  • Data analysis using standard curves constructed from serially diluted plasmids containing target sequences

  • Normalization to housekeeping genes (L32 or 18S)

• Northern Blot Hybridization: This technique has been successfully used to quantify VEGF mRNA expression in rat islet tissue under different experimental conditions, including measuring the 3.7-fold increase after hypoxic exposure .

• In Situ Hybridization: This approach allows visualization and quantification of VEGF mRNA expression in intact tissue sections, enabling spatial localization of expression patterns. It has been used to demonstrate the 4.6-fold increase in VEGF mRNA expression in cultured islet tissue over time .

What are the validated functional assays for measuring VEGF activity in rat samples?

Several functional assays have been validated for measuring VEGF activity in rat samples:

• Endothelial Cell Proliferation Assay: This assay measures the proliferative response of human umbilical vein endothelial cells (HUVEC) to rat VEGF164. Research has demonstrated that recombinant rat VEGF164 stimulates HUVEC proliferation in a dose-dependent manner, and this proliferation can be neutralized by increasing concentrations of anti-rat VEGF164 antibody. The neutralization dose (ND50) is typically 0.2-0.6 μg/mL in the presence of 20 ng/mL recombinant rat VEGF164 .

• Three-Dimensional In Vitro Angiogenesis Model: This assay evaluates endothelial cell invasion and capillary morphogenesis in response to VEGF stimulation. It has been used to demonstrate the bioactivity of VEGF derived from rat insulinoma RINm5F-2A cells .

• Conscious Cystometry: While not directly measuring VEGF activity, this functional assay has been used to assess the effects of VEGF/VEGFR2 signaling blockade on bladder function in rats. The methodology involves catheter implantation, saline infusion at 166.7 μl/min, and measurement of various urodynamic parameters including minimum pressure, threshold pressure, maximum micturition pressure, intermicturition interval, infused volume, void volume, and nonvoiding bladder contractions .

How can VEGF expression be modulated in rat models for therapeutic research?

VEGF expression can be modulated in rat models through several research-validated approaches:

• Hypoxic/Anoxic Conditioning: Exposing tissues to hypoxic/anoxic conditions for 8 hours has been demonstrated to increase VEGF mRNA expression 3.7-fold in rat islet tissue . This approach could potentially be used to upregulate VEGF expression before transplantation procedures.

• Culture Duration Manipulation: Extended culture of rat islet tissue leads to a significant 4.6-fold increase in VEGF mRNA expression over 7 days . This suggests that controlled culture conditions could be used to modulate VEGF expression for therapeutic applications.

• Pharmacological VEGFR2 Blockade: Administration of selective VEGFR2 tyrosine kinase inhibitors, such as Ki-8751 at 1 mg/kg, can effectively block VEGF signaling through VEGFR2 . This approach has demonstrated functional effects on bladder capacity and void volume in rat models.

• Targeted Antibody Neutralization: Using specific antibodies like Goat Anti-Rat VEGF164 Antigen Affinity-purified Polyclonal Antibody can neutralize VEGF activity in a dose-dependent manner, providing another approach for modulating VEGF signaling .

What are the implications of VEGF research in rat models for human inflammatory conditions?

VEGF research in rat models provides valuable insights into human inflammatory conditions:

• Biomarker Potential: Research demonstrates that VEGF expression changes significantly during inflammatory conditions in rat models, suggesting that VEGF may serve as a biomarker for analogous human inflammatory diseases. Specifically, findings suggest that VEGF may be a biomarker for interstitial cystitis/bladder pain syndrome in humans .

• Therapeutic Target Identification: The functional effects of VEGF/VEGFR2 blockade on bladder capacity and void volume in rat models of cystitis suggest that targeting this signaling pathway may have therapeutic potential for human inflammatory bladder conditions. Research indicates that "targeting VEGF/VEGFR2 signaling may be an effective treatment" for conditions such as interstitial cystitis/bladder pain syndrome .

• Sex-Specific Therapeutic Approaches: The observed sex differences in VEGF/VEGFR2 signaling responses in rat models suggest that sex-specific therapeutic approaches may be necessary for optimal treatment of inflammatory conditions in humans. Specifically, research suggests that "blockade of VEGF/VEGFR2 signaling has a greater effect on male mice with chronic CYP-induced bladder inflammation" .

How does inflammation alter the balance between pro-angiogenic and anti-angiogenic VEGF isoforms in rat tissues?

Inflammation induces complex changes in the balance between pro-angiogenic and anti-angiogenic VEGF isoforms in rat tissues:

• Upregulation of Multiple Isoforms: Research demonstrates that both VEGF-Axxxa (generally considered pro-angiogenic) and VEGF-Axxxb (generally considered anti-angiogenic) splice variants increase in rat lower urinary tract tissues following cyclophosphamide-induced cystitis .

• Tissue-Specific Expression Patterns: The relative expression of VEGF-Axxxa and VEGF-Axxxb isoforms varies across different tissue types, with VEGF-Axxxa predominating at the mRNA level in all examined lower urinary tract tissues .

• Transcriptional vs. Post-Transcriptional Regulation: Research suggests potential discrepancies between mRNA and protein expression patterns of VEGF splice variants, indicating that post-transcriptional modification may be an important mechanism regulating the balance between different VEGF isoforms during inflammatory conditions .

What are the key considerations for selecting VEGF antibodies for rat research?

When selecting VEGF antibodies for rat research, several technical considerations are critical:

• Isoform Specificity: Different antibodies may recognize specific VEGF isoforms. For example, certain antibodies have been validated for detecting rat VEGF164 . Researchers should select antibodies that specifically recognize the VEGF isoform(s) of interest for their particular application.

• Cross-Reactivity Profile: Understanding the cross-reactivity profile of VEGF antibodies is essential. For instance, certain anti-rat VEGF antibodies show significant cross-reactivity with mouse VEGF164 but less than 2% cross-reactivity with human VEGF-B, mouse VEGF-B, human VEGF-C, human VEGF-D, and mouse VEGF-D . This cross-reactivity information is crucial for experimental design and data interpretation.

• Application Validation: Antibodies should be validated for specific applications such as Western blot, immunohistochemistry, or neutralization assays. For example, Goat Anti-Rat VEGF164 Antigen Affinity-purified Polyclonal Antibody has been validated for detecting recombinant rat VEGF164 in Western blots and in immunohistochemistry of rat kidney sections .

• Neutralization Potential: For functional studies, antibodies with verified neutralizing activity should be selected. The neutralization dose (ND50) provides quantitative information about antibody potency. For example, certain anti-rat VEGF antibodies have an ND50 of 0.2-0.6 μg/mL in the presence of 20 ng/mL recombinant rat VEGF164 .

How can researchers optimize immunohistochemical detection of VEGF in rat tissues?

Optimizing immunohistochemical detection of VEGF in rat tissues involves several methodological considerations:

• Tissue Fixation and Processing: Proper fixation is critical for preserving VEGF antigenicity. Perfusion fixation followed by frozen sectioning has been successfully used for VEGF immunodetection in rat kidney .

• Antibody Concentration Optimization: Titration of primary antibody concentration is essential for optimal signal-to-noise ratio. For example, 15 μg/mL of Goat Anti-Rat VEGF164 Antigen Affinity-purified Polyclonal Antibody has been successfully used for immunohistochemistry of rat kidney sections .

• Incubation Conditions: Overnight incubation at 4°C may improve antibody binding and signal intensity, as demonstrated in protocols for VEGF detection in rat kidney sections .

• Detection System Selection: Appropriate secondary antibody and visualization systems should be selected based on the primary antibody species and desired sensitivity. For example, HRP-DAB cell and tissue staining kits have been successfully used for visualizing anti-rat VEGF antibody binding in kidney sections .

• Counterstaining: Appropriate counterstaining, such as hematoxylin, can provide tissue context for VEGF immunostaining .