# The Anti-angiogenic VEGF165b and VEGFR1 Signaling in Peripheral Artery Disease

> **NIH NIH R01** · AUGUSTA UNIVERSITY · 2021 · $673,667

## Abstract

Peripheral arterial disease (PAD) is caused by atherosclerosis that reduce blood flow, causing pain with
walking, amputation, a poor quality of life, and death. Current medical therapies in PAD have no effect on blood
flow. Vascular endothelial growth factor receptor (VEGFR)-2- mediated endothelial nitric oxide synthase
(eNOS) is the dominant pathway promoting hypoxia-dependent angiogenesis. Many pre-clinical studies have
focused on VEGFR2 for therapeutic angiogenesis but human studies have failed. VEGFR1+/- and mice with a
deleted VEGFR1 intracellular domain (tyrosine kinase dead, TK-/-) have impaired angiogenesis and perfusion
recovery compared to wild-type. The mechanisms by which VEGFR1 promotes hypoxia-dependent
angiogenesis are poorly understood and vastly understudied compared to VEGFR2. Alternative splicing of
VEGF-A results in a 6 amino acid switch that changes the pro-angiogenic VEGFxxxa (xxx = the number of
amino acids) to the “anti-angiogenic” VEGFxxxb (VEGF165b for 165 amino acids) isoform. Of critical importance
is our unexpected demonstration that the anti-angiogenic effects of the VEGFxxxb are directly linked to
VEGFR1 signaling; a finding that may shift the paradigm of hypoxia-dependent angiogenesis in general and
VEGFR signaling in PAD in particular. Using in vitro, preclinical, and human tissue studies, we showed that
VEGF165b is up-regulated with ischemia and preferentially binds to VEGFR1, limiting phosphorylation at Y1333.
A VEGF165b-specific inhibitory antibody increased hypoxia-dependent angiogenesis by increasing binding of
VEGF165a to VEGFR1, but the VEGF165b antibody had no effect on the binding of either VEGF165b or VEGF165a
to VEGFR2. With increased pVEGFR1Y1333 (but not VEGFR2 activation) and VEGFR1-pSTAT3 association,
and the angiogenic effects of anti-VEGF165b were VEGFR1-dependent. Both VEGF165a and VEGF165b bind
VEGFR1 with similar affinity but VEGF165b inhibited VEGF165a-mediated VEGFR1 activation at 10-fold lower
concentrations, despite being an equimolar activator of VEGFR2. We show VEGF165b induces an
inflammatory, anti-angiogenic M1 phenotype whereas anti-VEGF165b induces a reparative, pro-angiogenic M2
phenotype. VEGF165b+ cells were up-regulated in human monocytes found in PAD patients. Taken together,
these findings point to critical but previously unrecognized pro-angiogenic roles for VEGFR1 that are dictated
by VEGF165b. We pose that the anti-angiogenic effects of VEGF165b in hypoxia and PAD are mediated by
inhibition of VEGFR1 signaling in both ECs and macrophages. Aim 1 will determine whether antibody-
mediated VEGF165b inhibition induces hypoxia-dependent angiogenesis via a distinct VEGFR1-dependent
pathway. Aim 2 will determine the mechanisms by which VEGF165b promotes VEGFR1-mediated M1
macrophage polarization in mouse bone marrow derived macrophages (mBMDM) and human, peripheral
blood mononuclear cells. Aim 3 will determine the VEGFR1-mediated signaling pathways that promote
hypoxia-dependent angi...

## Key facts

- **NIH application ID:** 10071194
- **Project number:** 5R01HL141325-03
- **Recipient organization:** AUGUSTA UNIVERSITY
- **Principal Investigator:** BRIAN H ANNEX
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $673,667
- **Award type:** 5
- **Project period:** 2019-12-15 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10071194

## Citation

> US National Institutes of Health, RePORTER application 10071194, The Anti-angiogenic VEGF165b and VEGFR1 Signaling in Peripheral Artery Disease (5R01HL141325-03). Retrieved via AI Analytics 2026-06-14 from https://api.ai-analytics.org/grant/nih/10071194. Licensed CC0.

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