VEGF ₁₆₅ b Modulates Endothelial VEGFR1–STAT3 Signaling Pathway and Angiogenesis in Human and Experimental Peripheral Arterial Disease

Abstract

Rationale Atherosclerotic-arterial occlusions decrease tissue perfusion causing ischemia to lower limbs in patients with peripheral arterial disease (PAD). Ischemia in muscle induces an angiogenic response but the magnitude of this response is frequently inadequate to meet tissue perfusion requirements. Alternate splicing in the exon-8 of vascular endothelial growth factor (VEGF)-A results in production of pro-angiogenic VEGFxxxa isoforms (VEGF165a, 165 for the 165 amino acid product) and anti-angiogenic VEGFxxxb (VEGF165b) isoforms. Objective The anti-angiogenic VEGFxxxb isoforms are thought to antagonize VEGFxxxa isoforms and decrease activation of VEGF-Receptor-2 (VEGFR2), hereunto considered the dominant receptor in post-natal angiogenesis in PAD. Our data will show that VEGF165b inhibits VEGFR1-Signal Transducer and Activator of Transcription (STAT)-3 signaling to decrease angiogenesis in human and experimental PAD. Methods and Results In human PAD vs. control muscle-biopsies, VEGF165b: a) is elevated, b) is bound higher (vs. VEGF165a) to VEGFR1 not VEGFR2, and c) levels correlated with decreased VEGFR1, not VEGFR2, activation. In experimental PAD, delivery of an isoform specific monoclonal antibody (Ab) to VEGF165b vs. control-Ab enhanced perfusion in animal model of severe PAD (Balb/c strain) without activating VEGFR2-signaling but with increased VEGFR1-activation. Receptor pull-down experiments demonstrate that VEGF165b-inhibition vs. control increased VEGFR1-STAT3 binding and STAT3-activation, independent of janus activated kinase (Jak1)/Jak2. Using VEGFR1+/− mice that could not increase VEGFR1 after ischemia, we confirm that VEGF165b decreases VEGFR1-STAT3 signaling to decrease perfusion. Conclusions Our results indicate that VEGF165b prevents activation of VEGFR1-STAT3 signaling by VEGF165a and hence inhibits angiogenesis and perfusion recovery in PAD muscle.

Systems Pharmacology of VEGF165b in Peripheral Artery Disease

“…We also compared these model predictions to murine HLI; whereas tissue VEGF levels increase substantially in HLI, the model accurately captures trends in VEGF binding to VEGFR2 and the experimentally observed lack of increase (nonsignificant decrease) in VEGFR2 phosphorylation ( Figure d ). This suggests that, although there are many differences between human PAD and murine HLI (e.g., geometric scaling, sVEGFR1 expression, and time‐scale), receptor‐level signaling seem to be similar in this case (as supported by the recent work of Ganta et al …”

“…To capture the role of VEGF 165b in PAD, we incorporated: (1) its measured binding properties ( Figure b ); (2) changes in expression of VEGF 165a and VEGF 165b in skeletal muscle; and (3) secretion of VEGF 165b into blood (e.g., by monocytes). In the tissue compartments, we screened the possible range of relative VEGF 165a and VEGF 165b expression, maintaining constant free VEGF levels in plasma to mimic the roughly unchanged total VEGF protein and free VEGF in interstitial fluid in human PAD ( Supplementary Figure S2e, Supplementary Model Fitting ). In the bloodstream, we then increased secretion of VEGF 165b to capture the roughly threefold higher observed serum VEGF in patients with PAD than healthy humans .…”

“…We aim to develop a platform that can be used to screen potential pro‐angiogenic therapies for PAD. In achieving these objectives, we improve greatly on a previous pharmacokinetic/pharmacodynamic model of PAD, which was unable to capture the signaling impairment observed in PAD, by incorporating separate simulation of VEGFR2 ligand‐binding and site‐specific phosphorylation, and by incorporating recent discoveries about VEGF 165b ( Figure b ) and its relevance to PAD . By iteratively building upon and validating our models using both previously unpublished and published data, in vitro and in vivo , we improve the predictive capabilities without adding many parameters at a time.…”

A MicroRNA93–Interferon Regulatory Factor-9–Immunoresponsive Gene-1–Itaconic Acid Pathway Modulates M2-Like Macrophage Polarization to Revascularize Ischemic Muscle

“…α-SMA immunostaining in the ischemic adductor-muscle (IAM) from miR106b-93-25 −/− vs. WT showed significantly lower (~2X) arteriolar density (%average of α-SMA + vascular structures >50μm/image, p=0.002, Fig-1B). Furthermore, immunostaining of α-SMA and CD31 (%average of CD31 + cells/muscle fiber area (29) ) in ischemic gastrocnemius-muscle (IGA) from miR106b-93-25 −/− vs. WT showed a significant decrease in arteriogenesis (~3X, p=0.0001, Fig-1C) and angiogenesis (p>0.03, Fig-1D). …”

“…Unilateral femoral artery ligation and excision was performed on age and sex matched 12–16 week animals (22,23,24) .…”