WRW4, a selective LXA₄ receptor antagonist, reversed wound healing properties of LXA₄-MS.

<p>(A) qRT-PCR analysis was performed to assess the LXA₄ receptor <i>ALX</i> mRNA’s abundance in skin ulcers collected on days 2, 7, and 14 in the control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups. Data represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05), which is indicated as follows: *, LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS <i>vs</i>. Un-MS; and ^$, LXA₄-MS <i>vs</i>. soluble LXA₄. (B) Representative images of 1.5 cm dorsal wounds at days 2 and 7, with day 0 images serving as pre-injury images, are presented for the following groups: WRW4 (25 μl per animal—from a main peptide solution of 1 mg/ml), WRW4 + LXA₄-MS (WRW4 applied 10 minutes before MS application– 10 mg of LXA₄-MS), and LXA₄-MS (10 mg). (C) Wound healing index values for the groups outlined in (B). Index values range from 0 to 1, where a value of 0 indicates the original wound, and a value of 1 represents a completely closed wound. Data represent means ± SEM (n = 9 ulcers/group); One-way ANOVA was done to determine statistical significance (*<i>p</i> < 0.05).</p

Scanning electron microscopy (SEM) of microparticles and <i>in vitro</i> release of LXA₄ from MS.

<p>Representative images (2,000×) of (A) Unloaded and (B) LXA₄-MS morphologies. (C) <i>In vitro</i> cumulative release of LXA₄ from LXA₄-MS. LXA₄ concentration was determined by mass spectrometry over 48 h. Data are representative of two batches.</p

LXA₄-MS increased collagen deposition and angiogenesis and affected VEGF production.

<p>(A) Collagen deposition was measured using the ImageJ software with the Color Deconvolution plug-in, which measured densitometry in at least 12 random 400× fields in all groups, at days 2, 7 and 14. Percentages of collagen deposition are represented as means ± SEM (n = 6 ulcers/group). One-way ANOVA was used to determine statistical significance (<i>p</i> < 0.05) and is indicated as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^{, LXA₄-MS vs. soluble LXA₄. (B) Photomicrographs of wounds stained with Picro Sirius Red (200×) show collagen deposition at days 2, 7, and 14. (C) Histogram showing quantitative analysis of vascular density using the ImageJ software with the Cell Counter plug-in on 200× images. One-way ANOVA was performed to determine statistical significance (p < 0.05), which is indicated as follows: *, significant VEGF increase as compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS vs. Vehicle (PBS/glue);}, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄. (D) VEGF was quantified in all groups at days 2, 7 and 14 (represented as bars) in wounds via ELISAs as proxy to blood vessel density. Values are means ± SEM (n = 6 wounds/group). One-way ANOVA was performed to determine statistical significance (<i>p</i>< 0.05), which is indicated as follows: *, significant VEGF increase as compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^$, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

LXA₄-MS modulated cytokines generation in the skin.

<p>Skin ulcer tissues collected on days 0, 2, 7, and 14 from the control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups were homogenized to assess (A) IL-1β, (B) TNF-α, (C) IL-6, and (D) TGF-β production by ELISAs. Data (represented as bars) represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05) and indicated as follows: *, demonstrated significant differences compared to normal tissues (dashed line); ^#, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^$, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

Topical application of LXA₄-MS to skin ulcers accelerated wound closure and attenuated neutrophil chemotaxis.

<p>(A) Representative images of 1.5 cm dorsal wounds were collected on days 0, 2, 7, and 14 for the following groups: control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS. (B) Wound healing index values for the groups outlined in (A). Index values range from 0 to 1, where a value of 0 indicates the original wound, and a value of 1 represents a completely closed wound. Values are means ± SEM (n = 10 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05), which is as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^{, LXA₄-MS vs. soluble LXA₄. (C) ImageJ software was used to count inflammatory cells on day 2 in at least 12 random optical 400× fields per group. (D) Neutrophil accumulation (represented as bars) as measured by MPO. Values are means ± SEM (n = 5 wounds/group). One-way ANOVA was done to determine statistical significance (p < 0.05), which is indicated as follows: *, demonstrated significant increase compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS vs. Vehicle (PBS/glue);}, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and &, LXA₄-MS <i>vs</i>. soluble LXA₄. (E) qRT-PCR was performed to assess <i>MMP8</i> mRNA transcript abundance in skin ulcers collected on days 2, 7, and 14 from the vehicle (PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups. Data represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05) and indicated as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^$, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

Lipoxin A₄ encapsulated in PLGA microparticles accelerates wound healing of skin ulcers

<div><p>Lipoxin A₄ (LXA₄) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA₄ in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA₄-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA₄-MS, unloaded microparticles (Un-MS), soluble LXA₄, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA₄-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA₄, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA₄-MS reduced IL-1β and TNF-α, but increased TGF-β, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA₄-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (<i>MMP8</i>) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA₄-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA₄ receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA₄ must interact with ALX to induce wound healing. Our results show that LXA₄-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers.</p></div

LXA₄-MS increased macrophages and IL-4 production.

<p>(A) NAG (OD values) was quantified in skin ulcers collected on days 2, 7, and 14 from the vehicle (PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups (n = 5 ulcers/ group). (B) ELISAs were used to measure IL-4 production in skin ulcers collected on day 14 from the vehicle (PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups. Basal levels were also assessed. Data represent means ± SEM (n = 5 ulcers/ group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05) and indicated as follows: *, demonstrated significant differences compared to normal tissues (dashed line); ^#, LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^$, LXA₄-MS <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄.</p