Novel Anti-Microbial Peptide SR-0379 Accelerates Wound Healing via the PI3 Kinase/Akt/mTOR Pathway

<div><p>We developed a novel cationic antimicrobial peptide, AG30/5C, which demonstrates angiogenic properties similar to those of LL-37 or PR39. However, improvement of its stability and cost efficacy are required for clinical application. Therefore, we examined the metabolites of AG30/5C, which provided the further optimized compound, SR-0379. SR-0379 enhanced the proliferation of human dermal fibroblast cells (NHDFs) via the PI3 kinase-Akt-mTOR pathway through integrin-mediated interactions. Furthermore SR-0379 promoted the tube formation of human umbilical vein endothelial cells (HUVECs) in co-culture with NHDFs. This compound also displays antimicrobial activities against a number of bacteria, including drug-resistant microbes and fungi. We evaluated the effect of SR-0379 in two different would-healing models in rats, the full-thickness defects under a diabetic condition and an acutely infected wound with full-thickness defects and inoculation with <i>Staphylococcus aureus</i>. Treatment with SR-0379 significantly accelerated wound healing when compared to fibroblast growth factor 2 (FGF2). The beneficial effects of SR-0379 on wound healing can be explained by enhanced angiogenesis, granulation tissue formation, proliferation of endothelial cells and fibroblasts and antimicrobial activity. These results indicate that SR-0379 may have the potential for drug development in wound repair, even under especially critical colonization conditions.</p></div

<i>In vitro</i> activities of SR-0379 against Gram-positive and Gram-negative bacteria and fungi.

<p>The scores indicate the MICs (mg/ml) for gram-positive and gram-negative bacteria and fungi. MICs represent the individual data from two independent experiments.</p><p>NT: Not tested.</p

Cellular function of SR-0379.

<p>A) Effect of SR-0379 on NHDFs proliferation. NHDFs were treated with SR-0379 (1, 3 and 10 μg/ml) or FGF2 (0.1 μg/ml). N = 4 per group. *P<0.05 vs. control. B) The upper panel shows representative pictures of tube formation in a co-culture of HUVECs and NHDFs (Control, FGF2: 0.2 μg/ml) and SR-0379 (10 μg/ml). The lower panel shows the effects of SR-0379 on tube formation in a co-culture of HUVECs and NHDFs. N = 5 per group. *P<0.05, **P<0.01 vs. control. C) The upper panel shows representative pictures of the migration induced by FGF2 (0.1 μg/ml) and SR-0379 (10 μg/ml). The lower panel shows the effects of FGF2 (0.1 μg/ml) and SR-0379 (1 and 10 μg/ml) on migration. N = 4 per group. *P<0.05, **P<0.01 vs. control, #P<0.01 vs. SR-0379 (1 μg/ml). D) The upper panel shows representative pictures of the fibroblast-collagen-matrix contraction assay with FGF2 (0.3 μg/ml) and SR-0379 (1, 3, 10 and 30 μg/ml). The lower panel shows the effects of FGF2 (0.3 μg/ml) and SR-0379 (1, 3, 10 and 30 μg/ml) on fibroblast-collagen matrix contraction. N = 3 per group. *P<0.05, **P<0.01 vs. control.</p

Lead optimization from the angiogenic peptide AG30/5C.

<p>A) Major metabolites of AG30/5C determined by MALDI-TOF MS. The parent compound (AG30/5C) was incubated with rat serum <i>in vitro</i> 60 minutes. The metabolites were identified by comparison with the pre-incubation peptide. B) Sequences and net charges of AG30/5C and AG30/5C-derived peptides (SR-0007 and SR-0379). The lysine (K) of SR-007 was replaced with D-lysine in SR-0379. C) Effect of AG30/5C (10 μg/ml) and SR-0007 (10 μg/ml) on HUVECs proliferation. N = 3 per group. *P<0.05 vs. control. D) Effect of AG30/5C (10 μg/ml) and SR-0007 (10 μg/ml) on tube formation. The formation of capillary-like structures was observed in co-cultures of HUVECs and NHDFs. N = 5-12 per group. *P<0.05 vs. control. E) Stability of SR-0007 and SR-0379 in rat and human sera. SR-0007 and SR-0379 were quantified before or after incubation <i>in vitro</i> with rat and human sera for either 3 or 10 minutes. N = 2.</p

Effect of Ang II vaccine on Ang II-induced hypertension.

<p>(A) Systolic BP at steady state and under Ang II infusion on day 49. The control mice were immunized with 1 mg KLH. The experimental mice were immunized with 100ng or 1,000 ng Ang II-KLH. All groups contained 6 to 8 mice. The data are expressed as the mean systolic BP ± standard error (SE) of the mean. †<i>P</i><0.01 *<i>P</i><0.05. (B) The correlation between systolic BP under Ang II infusion and the anti-Ang II antibody titers in the sera of immunized mice. The titer is expressed as the dilution of serum giving half-maximal binding (optical density: OD 50%).</p

Histochemical analysis of kidney and heart after vaccination.

<p>(A and B) Kidney (upper panel) or heart with coronary artery (lower panel) was stained with H&E in control (saline) or immunized mice (1,000 ng Ang II-KLH with adjuvant) . The scale bar represents 100 µm in the upper panel or 20 µm in the lower panel. The results were examined (A) at day 42 after vaccination and (B) after Ang II infusion (day 56 after vaccination).</p

Conceptual schematic of the experiment.

<p>(A)Immunization step (Ang II-KLH: an antigen) (Step1) The antigen presenting cells (APCs) phagocytose the Ang II-KLH conjugate and present a T cell epitope of KLH to T cells through the major histocompatibility complex (MHC). T cells recognize it through T cell receptors and become activated. (Step2) B cells specific to Ang II (pentagons) differentiate to plasmacytes and proliferate with the help of activated T cells. Then, B cells produce anti-Ang II antibody. (B)Post-Immunization step (response to Ang II) (Step1) The APCs do not present the T cell epitope of Ang II to T cells. Therefore, T cells do not recognize it and are not activated by Ang II. (Step2) B cells specific to Ang II (pentagons) differentiate and proliferate in response to Ang II. Therefore, Ang II does not stimulate the production of anti-AngII antibody.</p

Effect of Ang II vaccine on SHR.

<p>A) Experimental protocol is shown. Ang II-KLH (Ang II-KLH group, n = 5) or saline (saline group, n = 5) were injected on days 0, 14, and 21 in twenty-four-week old male SHR. The anti-Ang II antibody titer and systolic BP were measured on days 0, 14, and 28. The Ang II-KLH group rats were immunized with 5 µg Ang II-KLH with CFA on day 0 and with 5 µg Ang II-KLH with IFA on day 14 and 21. B) Anti-Ang II antibody titers in the sera of immunized rats on days 0, 14, 28, and that of saline injected rats on day 28 were examined. *<i>P</i><0.01 vs. day 0. C) Systolic BP on days 0, 14, and 28 were shown. The data are expressed as the mean systolic BP ± standard error (SE) of the mean. *<i>P</i><0.05. vs. Saline.</p

Activation of the PI3 kinase/AKT/mTOR pathway by SR-0379 in NHDFs.

<p>A) Effects of SR-0379 on phosphorylated FAK (Tyr397 and Tyr925) and phosphorylated Akt (Ser473) as determined by Western blot. The cells were treated with SR-0379 (10 μg/ml) for 0, 5, 15 and 30 minutes or LL-37 (10 μg/ml) for 15 minutes. B) Effects of SR-0379 on phosphorylated FAK (Tyr397 and Tyr925) and phosphorylated Akt (Ser473) as determined by Western blot. The cells were treated with SR-0379 (0.1, 0.3, 1, 3 and 10 μg/ml) for 30 minutes. C, D) Effects of RGD peptide and wortmannin on the SR-0379-induced phosphorylation of FAK (Tyr397 and Tyr925) and Akt (Ser473) as determined by Western blot. The cells were preincubated with RGD peptide (30, 100 and 300 μM, an inhibitor of integrin-ligand interactions) (C) or wortmannin (100 nM) (D) for 30 minutes and were then treated with SR-0379 (10 μg/ml) for 30 minutes. E) Effects of RGD, wortmannin and rapamycin on the NHDFs proliferation stimulated by SR-0379. The cells were preincubated with RGD (1000 μM), wortmannin (100 nM) or rapamycin (1 nM) for 2 hours and then were treated with SR-0379 (10 μg/ml). N = 3 per group. **P<0.01 vs. control, ## P<0.01 vs. SR-0379 (10 μg/ml). F) Effects of Akt knockdown using siRNA on the NHDFs proliferation stimulated by SR-0379. The cells were pretreated with Akt si RNA or Control si RNA for 24 hours and then were treated with SR-0379 (1, 3 and 10 μg/ml). N = 4 per group. **P<0.01 vs. control si RNA, # P<0.05 vs. SR-0379 (1 μg/ml) treated with control siRNA.</p

Effects of SR-0379 and FGF2 on full-thickness wound model with flap in diabetic rat model.

<p>A) Representative pictures of skin flaps in the streptozotocin-induced diabetic model in the saline (control), SR-0379 (0.2 mg/ml) and FGF2 groups (0.06 mg/ml) on days 0, 6, 13 and 20. B) Quantification of the wound area is represented as a percentage of the initial wound area. N = 6 per group. **P<0.01 vs. control, ##P<0.01 vs. FGF2. C) Days to complete healing by the contraction of full-thickness skin flaps in the streptozotocin-induced diabetic model.</p