Effects of L-Ornithine-L-Aspartate on Angiogenesis and Perfusion in Subacute Hind Limb Ischemia: Preliminary Study

Abstract

The current treatment options for peripheral arterial disease (PAD) are limited due to a lack of significant high-level evidence to inform clinical decisions and unfavorable outcomes in terms of cost-effectiveness and amputation rates. In order to suggest the use of the commercially available L-Ornithine-L-Aspartate (LOLA) for treating PAD, we induced hind limb ischemia (HLI) by unilaterally ligating the femoral artery in a rat model. The rats were randomly divided into three groups, with seven rats assigned to each group: group 1 (control), group 2 (sorbitol), and group 3 (LOLA). Intraperitoneal injections were administered five times on post-operative days (PODs) 3, 5, 7, 10, and 12. Perfusion imaging was conducted on PODs 7 and 14 and compared to pre-operative perfusion imaging. Immunohistochemistry staining and Western blotting were performed after the final perfusion imaging. Group 3 showed a significant increase in perfusion, high CD31-positive capillary lumen density, and substantial overexpression of VEGF in the ischemic limb during the subacute phase of HLI. In conclusion, this study provides the first documented evidence of angiogenesis and perfusion recovery in the subacute phase of the HLI model following the administration of LOLA. With LOLA readily available on the commercial market, the implementation of LOLA treatment for PAD in humans can be expedited compared to other therapies still in the developmental stage.

Keywords: L-ornithine-L-aspartate; angiogenesis; peripheral arterial disease.

Figure 1

Change in perfusion after single ligation of proximal femoral artery in HLI model. (a) Schematics of the surgery. Rats underwent unilateral right femoral artery ligation as close as possible to the inguinal ligament (b). Laser Doppler perfusion imaging. Regions of interest (ROI) were drawn around feet and ankles, and perfusion ratios were calculated. (c) A point in linear plot of experiments representing percentage of perfusion recovery (n = 2). (d) Schematics of the protocol. After pre-operative perfusion imaging and surgery, substances corresponding to each group were injected intraperitoneally five times within two weeks of surgery. Perfusion imaging was scanned at PODs 7 and 14, followed by sampling of gastrocnemius muscles of ischemic limbs. HLI: hind limb ischemia; pre-op: pre-operative; POD: post-operative day.

Figure 2

Perfusion recovery after administration of LOLA. (a) Laser Doppler perfusion imaging of three groups. Regions of interest (ROI) were drawn around feet and ankles, and perfusion ratios were calculated. Red represents the highest flow, and blue represents the lowest. (b) Bar plots of experiments representing percentage of perfusion recovery in three groups (group 1, blue; group 2, red; group 3, green). Error bar indicates standard error of mean. * indicates significant difference at p < 0.05. Group 1 (control; n = 7); group 2 (sorbitol; n = 7); group 3 (LOLA; n = 7); LOLA: L-Ornithine-L-Aspartate; POD: post-operative day.

Figure 3

Angiogenesis after administration of LOLA. (a) Protein expression of CD 31 was detected by immunohistochemistry in gastrocnemius muscles of HLI rats. The scale bars indicate 2000 µm. (b) High magnification of (a). The scale bars indicate 100 µm. (c) CD31 positive cell density. Error bar indicates standard error of mean. *** indicate significant difference at p < 0.001. Group 1 (control; n = 7); group 2 (sorbitol; n = 7); group 3 (LOLA; n = 7); LOLA: L-Ornithine-L-Aspartate.

Figure 4

Angiogenesis after administration of LOLA. (a) Protein expression of VEGF was detected by immunohistochemistry in gastrocnemius muscles of HLI rats. The scale bars indicate 2000 µm. (b) High magnification of (a). The scale bars indicate 100 µm. (c) Western blotting of VEGF. Relative expression of a VEGF homodimer (46 kDa) of 23 kDa subunits was normalized to endogenous control GAPDH. Western blotting was incidentally performed on samples obtained from one rat of the control group (∆), and expression level was estimated from the initial result. Although Western blotting and some other analyses were conducted in a sham, the results are not included in this report. (d) VEGF expression level. Error bar indicates standard error of mean. * indicates significant difference at p < 0.05, respectively. Group 1 (control; n = 7); group 2 (sorbitol; n = 7); group 3 (LOLA; n = 7); GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; LOLA: L-Ornithine-L-Aspartate; VEGF: vascular endothelial growth factor.