Immunohistochemistry showing longitudinal changes in expression of STAT4 in diabetic db/db and heterozygous control mice.

<p><b>(A)</b> Representative micrographs showing STAT4 immunostaining (red) and nuclear staining using DAPI (blue) of formalin fixed paraffin embedded whole wounds of db/db-/- and control mice at the time of wounding (T0) and after 8 and 48 hours, respectively. Magnification: 100x; (B) High power (400x) images of the dermis (top) and adipose tissue (bottom) layers of the dbdb-/- and control mice at 48 hours post-wounding. Solid arrows indicate peri-nuclear localization of STAT4 and dashed arrows indicate nuclear localization. Insets represent higher magnification of the nuclear (top) or peri-nuclear (bottom) localization of the signal. (C) Grading of STAT4 staining in the dermis and dermal adipose tissue of diabetic db/db and control mice. A scale of “0” to”3” was used to quantify the abundance of staining. A number of 5–8 micrographs/section from n = 3 mice/group were graded by 4 independent observers in a blinded manner. * P ≤0.05.</p

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

<div><p>Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.</p></div

Pegylated versions of PA inhibit complement activity in a hemolytic assay.

<p>(A) Hemolytic assays using factor B-depleted serum were performed with PA dissolved in DMSO and its pegylated derivatives dissolved in water. Factor B-depleted serum was incubated with 0.77mM of each peptide and then added to sensitized sheep erythrocytes. (B) Titration of increasing amounts of PA and PA-dPEG24 in the hemolytic assay. Water and DMSO were used as vehicle controls in the presence of factor B-depleted serum. GVBS⁺⁺ is a buffer- only control. Values are the means of three independent experiments. Error bars represent the SEM.</p

PA-dPEG24 inhibits complement activation <i>in vivo</i>.

<p>(A) Rats were IV administered PA-dPEG24 at 10 (n = 2) or 20mg/mL (n = 2) in 0.9% NaCl or received a vehicle control (n = 2) or were sham treated (n = 2). B, Rats were IV administered PA-dPEG24 at 10mg/mL (n = 4), 20mg/mL (n = 4) or 30mg/mL (n = 4) in 10mM Na₂HPO₄, 0.9% NaCl or received a vehicle control (n = 3). At the indicated time points post-administration, blood was collected and plasma isolated. Plasma was tested in hemolytic assays with human AB erythrocytes. Error bars represent the SEM.</p

Linear mode MALDI-TOF mass spectrometry analysis of PA-dPEG24 oligomeric state.

<p>PA-dPEG24 in acidified water was analyzed by linear mode mass spectrometry. The spectrum shows two peaks for PA-dPEG24 with a monomer peak at 2778 and a dimer form at 5553.</p

PA binds to MBL and ficolins and does not displace C1s from C1q.

<p>(A) The AcPA and CP2 peptides were adsorbed to a microtiter plate and incubated with a constant amount of MBL or K55Q. Bound MBL was detected with a polyclonal goat anti-MBL sera followed by HRP-conjugated anti-goat sera. (B) The various ficolins indicated in the figure were adsorbed to the microtiter plate and incubated with biotinylated PA followed by a neutravidin conjugate. BSA was used as a negative control for binding. (C) Partially purified C1 was mixed with increasing amounts of CP or PA and added to a microtiter plate coated with monoclonal antibody to C1q. After washing, C1s signal present in intact C1 complexes was measured by ELISA using polyclonal antibody to C1s (CP, red line; PA, blue line) or polyclonal antibody to C1q (green line) to confirm C1q was bound to the plate. Data represent the means of three independent experiments. Error bars denote SEM.</p

Complement activation and cellular inflammation for acute skin wounds in diabetic mice.

<p>(A) C5a concentration in wound beds of diabetic and control mice absorbed by filter paper and assayed by ELISA. Left and right wounds averaged for n = 3 mice in each group and time point: 10 min (P = 0.05) 2h (P = 0.002), 4h (P = 0.001), 24h (P = 0.05). * P ≤0.05 vs. hetero (control). (B) C3-fragment deposition (C3 opsonization) in the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by immunofluorescence (n = 2). (C) Nucleated cell infiltration into the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by DAPI fluorescence (n = 2). Data are means ± SEM.</p

Analysis of leukocyte inflammation of wounds for diabetic mice treated with PIC1 at 14 days.

<p>Representative wound histology (H&E) for (A) control scaffold only mice, and (B) PIC1 impregnated skin scaffold. (C) Averaged inflammatory index of leukocytes (predominantly neutrophils) for db/db mice at day 14 post wounding for wounds treated with PIC1 complement inhibitor in a skin scaffold or control skin scaffold, n = 6 in each group. Data are means ± SEM. * P ≤0.05 vs. saline control.</p

Model of PA inhibition of C1/MBL function.

<p>Our data favors a model in which PIC1 derivatives functionally disrupt the C1s-C1r-C1r-C1s/MASP interaction with the CLR of C1q/MBL, respectively. By PIC1 binding to the serine protease interaction site on CLR, the serine proteases are hypothesized to be in a suboptimal conformation for autoactivation and initiation of the classical and/or lectin pathways of complement.</p

Longitudinal changes in gene expression of STAT4, Ccl2 and Ccr2 in diabetic db/db mice and controls.

<p>Wounds were excised at indicated time points and gene expression was measured by real-time PCR using Taqman probes. Results are expressed as 1/ΔCt and represent the average of two wounds/mouse from 3 mice/group +/-SEM. * P ≤0.05 vs. dbhet (control); # P ≤0.05 vs. t = 0 (approximately 10 minutes after wounding).</p