Neuropilin-1 (Nrp1) is a multifunctional transmembrane protein that is abundantly expressed on the surface of various cell types, where it binds to class 3 semaphorins (Sema3), heparan sulfate, and vascular endothelial growth factors (VEGFs). Nrp1 plays a crucial role in angiogenesis and vascular permeability regulation, particularly in the VEGF signaling pathway, where it acts as a co-receptor for VEGF receptor 2 (VEGFR2), modulating VEGFR2 activation and downstream signaling. However, the specific role of Nrp1 in VEGF-mediated vascular permeability remains controversial. Some studies suggest that Nrp1 positively regulates VEGF-mediated vascular permeability, while others indicate that it has no significant effect. This inconsistency may be due to differences in experimental models, tissue types, or experimental setups.

To clarify the specific role of Nrp1 in VEGF-mediated vascular permeability, this study constructed endothelial cell-specific Nrp1 knockout mice and investigated the regulatory mechanisms of Nrp1 in different tissues within the VEGF/VEGFR2 signaling pathway, revealing the tissue-specific role of Nrp1 in vascular permeability.

Source of the Paper

This paper was co-authored by Sagnik Pal, Yangyang Su, Emmanuel Nwadozi, Lena Claesson-Welsh, and Mark Richards, all from the Department of Immunology, Genetics, and Pathology at Uppsala University, Sweden. The paper was published on December 1, 2024, in the journal Angiogenesis, titled “Neuropilin-1 controls vascular permeability through juxtacrine regulation of endothelial adherens junctions.”

Research Process and Experimental Design

1. Construction and Validation of Endothelial Cell-Specific Nrp1 Knockout Mice

The study first constructed endothelial cell-specific Nrp1 knockout mice (Nrp1 iECKO) and validated the significant reduction of Nrp1 expression in endothelial cells through Western blot and immunofluorescence staining. To investigate the role of Nrp1 in VEGF-mediated vascular permeability, the research team employed various experimental methods, including intravital microscopy, Evans Blue dye extravasation assays, and Miles assays.

2. VEGF-Mediated Vascular Permeability Experiments

The research team assessed VEGF-induced vascular permeability in different tissues (e.g., ear skin, back skin, and trachea). Using intravital microscopy, they found that VEGF-induced vascular permeability was significantly increased in the ear skin of Nrp1 iECKO mice but significantly decreased in the back skin and trachea. These results indicate that Nrp1 has a tissue-specific role in VEGF-mediated vascular permeability.

3. Experiments with Global Nrp1 Knockout Mice

To further explore the role of Nrp1 in vascular permeability, the research team constructed global Nrp1 knockout mice (Nrp1 iKO). The experimental results showed that VEGF-induced vascular permeability was significantly reduced in the ear skin of global Nrp1 knockout mice, while the results in the back skin and trachea were consistent with those of Nrp1 iECKO mice. This suggests that the role of Nrp1 in vascular permeability is not only dependent on endothelial cells but may also be influenced by perivascular cells.

4. Expression of Nrp1 in Perivascular Cells

Through immunofluorescence staining, the research team found that Nrp1 expression was higher in perivascular cells in the ear skin but lower in the back skin. This finding suggests that the expression level of Nrp1 in perivascular cells may influence its role in VEGF-mediated vascular permeability.

5. Interaction Between Nrp1 and the VEGFR2 Signaling Pathway

The research team further explored the interaction between Nrp1 and the VEGFR2 signaling pathway. By constructing VEGFR2 Y949F mutant mice, they found that Nrp1 regulates VEGF-mediated vascular permeability in the ear skin by modulating the phosphorylation of the VEGFR2 Y949 site. In contrast, in the back skin and trachea, Nrp1 positively regulates VEGF-mediated vascular permeability through the VEGFR2 Y949 site.

Main Research Findings

1. Tissue-Specific Role of Nrp1 in VEGF-Mediated Vascular Permeability: In the ear skin, Nrp1 negatively regulates VEGF-mediated vascular permeability, while in the back skin and trachea, Nrp1 positively regulates VEGF-mediated vascular permeability.

2. Results from Global Nrp1 Knockout Mice: VEGF-induced vascular permeability was significantly reduced in the ear skin of global Nrp1 knockout mice, while the results in the back skin and trachea were consistent with those of endothelial cell-specific Nrp1 knockout mice.

3. Expression of Nrp1 in Perivascular Cells: Nrp1 expression was higher in perivascular cells in the ear skin but lower in the back skin, suggesting that the expression level of Nrp1 in perivascular cells may influence its role in VEGF-mediated vascular permeability.

4. Interaction Between Nrp1 and the VEGFR2 Signaling Pathway: Nrp1 regulates VEGF-mediated vascular permeability by modulating the phosphorylation of the VEGFR2 Y949 site, with different regulatory mechanisms observed in the ear skin and back skin.

Research Conclusions and Significance

This study reveals the tissue-specific role of Nrp1 in VEGF-mediated vascular permeability and elucidates the molecular mechanisms by which Nrp1 regulates vascular permeability through the VEGFR2 signaling pathway. The results indicate that Nrp1 negatively regulates VEGF-mediated vascular permeability in the ear skin to maintain vascular barrier stability, while it positively regulates VEGF-mediated vascular permeability in the back skin and trachea to promote vascular permeability. Additionally, the expression level of Nrp1 in perivascular cells significantly influences its role in VEGF-mediated vascular permeability.

The scientific value of this study lies in its revelation of the complex regulatory mechanisms of Nrp1 in vascular permeability, providing new perspectives for understanding the role of the VEGF signaling pathway in vascular biology. Furthermore, the research findings offer a theoretical basis for developing drugs targeting Nrp1 to regulate vascular permeability, with potential therapeutic applications.

Research Highlights

1. Tissue-Specific Role: This study is the first to reveal the tissue-specific role of Nrp1 in VEGF-mediated vascular permeability, providing new insights into the function of Nrp1 in different tissues.

2. Interaction Between Nrp1 and the VEGFR2 Signaling Pathway: The study elucidates the molecular mechanisms by which Nrp1 regulates VEGF-mediated vascular permeability through the phosphorylation of the VEGFR2 Y949 site.

3. Role of Perivascular Cells: The study reveals that the expression level of Nrp1 in perivascular cells significantly influences its role in VEGF-mediated vascular permeability, providing new clues for understanding the regulatory mechanisms of vascular permeability.

Other Valuable Information

This study also explores the potential role of Nrp1 in tumor angiogenesis and vascular permeability, offering new strategies for developing Nrp1-targeted tumor therapies. Additionally, the research findings provide a theoretical basis for understanding the role of Nrp1 in other vascular-related diseases, such as diabetic retinopathy and age-related macular degeneration.

Through systematic experimental design and in-depth data analysis, this study reveals the complex regulatory mechanisms of Nrp1 in VEGF-mediated vascular permeability, providing important scientific evidence for understanding the molecular mechanisms of vascular biology.