Background & Aims

Patients with peripheral arterial disease (PAD) often suffer from chronic pain symptoms including intermittent claudication. However, pathophysiology of peripheral ischemic pain in PAD has not been fully understood. Vascular endothelial growth factor (VEGF)-A, its receptor tyrosine kinase VEGFR-2 and co-receptor neuropilin (NRP)-1 originally known as key regulators of angiogenesis are up-regulated in ischemic muscles in patients with PAD. VEGF-A is by alternative RNA splicing of exon 8 in VEGF-A mRNA, generated as two isoforms represented by VEGF-A165a and VEGF-A165b. VEGF-A165a has binding affinity to VEGFR-1, -2 and NRP1, -2. While VEGF-A165b exhibits similar binding affinity to VEGFR-1, -2, and NRP-2, but not to NRP-1. We have previously showed that the relative levels of altering VEGF-A165a and VEGF-A165b are critical for developing muscle hyperalgesia in a rat model of PAD. In this study, we investigated the role of VEGFR-1, -2, and NRP-1, -2 on muscle hyperalgesia in this model.

Methods

Under deep anesthesia with an intraperitoneal injection of a mixture consisting of butorphanol, medetomidine and midazolam, both left common iliac and iliolumbar arteries were ligated respectively, through a midline laparotomy (PAD rats). Sham rats were exposed the arteries without ligation. Cutaneous blood flow in hindlimb was measured by laser Doppler flowmetry. Von Frey test and Plantar-test of the hindpaw and the Randall-Selitto test on the calf were performed. In addition, treadmill test was performed to measure walking distance as an assessment of intermittent claudication. Histological examination of both gastrocnemius muscle (GM) and skin over the muscle were performed on day 4 and 14 after arterial ligation. On day14, effects of both VEGFR-1 and -2 neutralizing antibody (R1 and R2 Neu-Ab) were evaluated on behavioral responses respectively. Furthermore, effects of both NRP-1 and -2 neutralizing antibody (NRP1 and NRP2 Neu-Ab) were also evaluated respectively.

Results

Muscle hyperalgesia of the GM was observed for 3 weeks, and reduced walking distance was observed for 12 weeks after ligation in PAD rats. Mechanical allodynia of the hindpaw was observed for 1 week although heat hyperalgesia was not. Reduced cutaneous blood flow was also observed for 6 weeks. Infiltrations of the inflammatory cells and pathological changes of muscle cells (e.g., necrotic cells, central cores) were seen in the GM on day 4 but recovered by day 14. On day 14, intramuscular injection of R1 Neu-Ab suppressed muscle hyperalgesia. By contrast, intramuscular injection of R2 Neu-Ab made muscle hyperalgesia worse in PAD rats. In addition, R2 Neu-Ab induced muscle hyperalgesia in sham rats. Furthermore, intramuscular injection of NRP1 Neu-Ab made muscle hyperalgesia worse in both PAD and sham rats, even though NRP2 Neu-Ab did not make any differences in both groups.

Conclusions

VEGF-A165a-VEGFR1 signaling plays a critical role for developing muscle hyperalgesia associated with PAD. On the other hand, both VEGF-A165a-NRP1-VEGFR1 and VEGF-A165b-VEGFR2 signaling would have a protective role for muscle hyperalgesia.

References

Hori et al. Altering VEGF-A165a/VEGF-A165b balance contribute to muscle hyperalgesia in a rat model of peripheral arterial disease. 19th world congress on Pain, PTU237, Abstract, 2022

Presenting Author

Kiyomi Hori

Poster Authors

Kiyomi Hori

PhD

Kanazawa University

Lead Author

Hiroaki Okuda

PhD

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Tatsuya Ishikawa

PhD

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Yoshitake Shiraishi

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Tsuneo Nakamura

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Illia Potapenko

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Zao Yuxue

PhD

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Noriyuki Ozaki

MD

Department of Functional Anatomy, Kanazawa University Graduate School of Medical Science

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology