Fibronectin-derived Epiviosamines enhance PDGF-BB-stimulated human dermal fibroblast migration in vitro and granulation tissue formation in vivo.

Fibronectin (FN) is a multimodular glycoprotein that is a critical component of the extracellular matrix (ECM) anlage during embryogenesis, morphogenesis, and wound repair. Our laboratory has previously described a family of FN-derived peptides collectively called "epiviosamines" that enhance platelet-derived growth factor-BB (PDGF-BB)-driven tissue cell survival, speed burn healing, and reduce scarring. In this study, we used an agarose drop outmigration assay to report that epiviosamines can enhance PDGF-BB-stimulated adult human dermal fibroblast (AHDF) outmigration in a dose-dependent manner. Furthermore, these peptides can, when delivered topically, stimulate granulation tissue formation in vivo. A thiol-derivatized hyaluronan hydrogel cross-linked with polyethyleneglycol diacrylate (PEGDA) was used to topically deliver a cyclized epiviosamine: cP12 and a cyclized engineered variant of cP12 termed cNP8 to porcine, full-thickness, excisional wounds. Both cP12 and cNP8 exhibited dose-dependent increases in granulation tissue formation at day 4, with 600 µM cNP8 significantly enhancing new granulation tissue compared to vehicle alone. In contrast to previous studies, this study suggests that epiviosamines can be used to increase granulation tissue formation without an exogenous supply of PDGF-BB or any cell-binding peptides. Thus, epiviosamine may be useful topically to increase granulation tissue formation in acute wounds.

Vasoactive effect of fibronectin-derived epiviosamine-1 and related peptides in quiescent and stress models.

OBJECTIVE: Following thermal burn injury, plasma fibronectin degrades within the interstitium; one possible product is EVA-1, PSHISKYILRWRPK found within the FNIII1 . EVA-1 ameliorates thermal burn injury progression, and binds to and enhances PDGF-BB in promoting cell metabolism, growth and survival; shorter related peptides lose these abilities. Here we study the effect of EVA-1 and shorter peptides for their vasoactivity under quiescent and stress conditions. METHODS: Using the hamster cheek pouch intravital microscopy model, five EVA-1 related peptides were applied to small arterioles via micropipette (10-16 -10-4 mol L-1 ) in quiescent tissue and after defined stress: nitric oxide or heat. RESULTS: Peak dilation occurred with nanomolar doses (longer peptides) or below (shorter peptides), blocked by propranolol (beta-adrenergic receptor antagonist). Micromolar doses of the same peptides induced only constriction, not antagonized by phentolamine (alpha-adrenergic receptor antagonist). Scrambled variants of two peptides yielded only constriction, suggesting constriction might be due peptide charge. Each stressor caused a left shift in dilation response, blocked by carazolol. CONCLUSIONS: Thus, this important region of FNIII1 contains sequences that have a gradation of biological functions dependent on the length of the peptide sequence, with increased efficacy for dilation following stressors.

Blood vessel occlusion in peri-burn tissue is secondary to erythrocyte aggregation and mitigated by a fibronectin-derived peptide that limits burn injury progression.

Although vascular occlusion has long been noted in peri-burn tissue, the literature is inconsistent regarding the nature of the occlusion, with articles in the 1940s claiming that erythrocytes were the culprit and in the 1980s-1990s that microthrombi were responsible. To better define the nature of vessel occlusion, we studied two porcine burn models, a hot comb horizontal injury model and a vertical injury progression model. In both cases, tissue from the first two days after burn were stained with hemotoxylin and eosin, or probed for platelets or for fibrinogen/fibrin. Erythrocytes, identified as nonstained, clumped, anuclear, 5 µm cells, occluded most blood vessels (BVs) in both burn models. In contrast, platelet or fibrinogen/fibrin antibodies stained BV occlusions minimally at early time points, and only up to 16% of deep dermal BVs at 48 hours in the hot comb model and up to 7% at 24 hours in the vertical injury progression model. Treatment of animals with a fibronectin-derived peptide (P12), which limits burn injury progression and can dilate peripheral microvasculature, reduced erythrocyte occlusion by at least 50%, speeded healing and reduced scarring. Early erythrocyte aggregation, rather than thrombosis, explains the ineffectiveness of anticoagulants to prevent burn injury progression.

ICG angiography predicts burn scarring within 48 h of injury in a porcine vertical progression burn model.

The current standard of care in determining the need to excise and graft a burn remains with the burn surgeon, whose clinical judgment is often variable. Prior work suggests that minimally invasive perfusion technologies are useful in burn prognostication. Here we test the predictive capabilities of Laser Doppler Imaging (LDI) and indocyanine green dye (ICG) angiography in the prediction of burn scarring 28 days after injury using a previously validated porcine burn model that shows vertical progression injury. Twelve female Yorkshire swine were burned using a 2.5 × 2.5 cm metal bar at variable temperature and application times to create distinct burn depths. Six animals (48 injuries total) each were analyzed with LDI or ICG angiography at 1, 24, 48, and 72 h following injury. A linear regression was then performed correlating perfusion measurements against wound contraction at 28 days after injury. ICG angiography showed a peak linear correlate (r(2)) of .63 (95% CI .34 to .92) at 48 h after burn. This was significantly different from the LDI linear regression (p < .05), which was measured at r(2) of .20 (95% CI .02 to .39). ICG angiography linear regression was superior to LDI at all timepoints. Findings suggest that ICG angiography may have significant potential in the prediction of long-term burn outcomes.

Indocyanine green dye angiography accurately predicts survival in the zone of ischemia in a burn comb model.

INTRODUCTION: Surgical evaluation of burn depth is performed via clinical observation, with only moderate reliability. While perfusion analysis has been proposed to enhance accuracy, no perfusion study has attempted to predict burn extension into the area of ischemia surrounding the original insult. We examined whether laser Doppler imaging (LDI) and indocyanine green (ICG) angiography predicted survival in the zone of ischemia in a porcine hot comb burn model. METHODOLOGY: Six full-thickness wounds were created on 5 female Yorkshire swine using a validated porcine hot comb burn model. 4 full-thickness burns were created separated by 3 unburned interspaces that represent the zone of ischemia. The interspaces between each comb burn were monitored using LDI and ICG Angiography at 1, 4, 24, and 48 h after burn. Interspace survival was assessed via gross observation and blinded histological readings 7 days after injury. RESULTS: ICG Angiographic assessments of burn perfusion were significantly different in viable vs. non-viable interspace perfusion at 1 h, 4 h, and 48 h. Temporal plotting of a trend-line derived from quantitative perfusion measurements rendered two distinct graphs, allowing for the derivation of a predictive algorithm to separate viable and non-viable interspaces. LDI revealed no such prognostic trend. CONCLUSION: Results from a validated porcine burn comb model suggest that ICG angiography has significant potential in the prediction of burn progression early after burn. However, the full potential of this technology cannot be determined until completion of clinical trials.

Fibronectin peptides that bind PDGF-BB enhance survival of cells and tissue under stress.

Stressors after injury from a multitude of factors can lead to cell death. We have identified four fibronectin (FN) peptides: two from the first FN type III repeat (FNIII1), one from the 13th FN type III repeat (FNIII13), and one from FN variable region (IIICS), which when tethered to a surface acted as platelet-derived growth factor-BB (PDGF-BB) enhancers to promote cell survival. One of the FNIII1 peptides and its smallest (14-mer) bioactive form (P12) were also active in solution. Specifically, P12 bound PDGF-BB (KD=200 nM), enhanced adult human dermal fibroblast (AHDF) survival under serum starvation, oxidative or endoplasmic reticulum stressors, and limited burn-injury progression in a rat hot comb model. Furthermore, P12 inhibited endoplasmic reticulum stress-induced c-Jun N-terminal kinase (JNK) activation. Although many growth factors have been found to bind FN directly or indirectly, here we identify peptide sequences of growth factor-binding sites in FN. The finding of these peptides further delineated how the extracellular matrix protein FN can support cell survival. As the peptide P12 is active in either soluble form or tethered to a substrate, it will have multifactorial uses as a bioactive peptide by itself or in tissue engineering.

A fibronectin peptide redirects PDGF-BB/PDGFR complexes to macropinocytosis-like internalization and augments PDGF-BB survival signals.

Growth factor-binding domains identified in various extracellular matrix proteins have been shown to regulate growth factor activity in many ways. Recently, we identified a fibronectin peptide (P12) that can bind platelet-derived growth factor BB (PDGF-BB) and promote adult human dermal fibroblast (AHDF) survival under stress. In vivo experiments in a porcine burn injury model showed that P12 limited burn injury progression, suggesting an active role in tissue survival. In this report, we explored the molecular mechanism of this peptide in ADHF under nutrient deprivation. Our results showed that P12 acted like some cell-penetrating peptides in that it redirected ligand-bound PDGF receptor (PDGFR) from the clathrin-dependent endocytic pathway to a slower, macropinocytosis-like pathway. P12 slowed internalization and degradation of PDGF-BB, augmented its survival signals, and promoted cell survival after nutrient removal. Our findings demonstrate a mechanism for a potential therapeutic peptide that increases cell and tissue survival by acting as a cofactor to PDGF-BB.

High cell density attenuates reactive oxygen species: implications for in vitro assays.

In vitro cell-based assays are an essential and universally used step in elucidation of biological processes as well as in drug development. However, results obtained depend on the validity of protocols used. This statement certainly pertains to in vitro assays of oxidative stress. The holy grail of in vitro models is reliability and predictability of outcomes that relate to a single variable like addition of hydrogen peroxide or xanthine oxidase. Without such validated outcomes, comparison of results among different laboratories is not possible. Achieving this goal requires a thorough understanding of the complex interplay between the cells, their environment, and the experimental assays. Furthermore, as this knowledge is attained, it must be disseminated and used to update and standardize existing protocols. Here, we confirm and extend the effect of pyruvate and cell density on in vitro oxidative stress assays. Cell viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt (XTT) into a colored formazan dye. Extracellular hydrogen peroxide concentrations were measured using the foxp3 assay. We confirmed a previously reported finding that pyruvate, a common ingredient in cell culture media, acts as an extracellular scavenger of reactive oxygen species. We also demonstrated that cell density directly correlates with resistance to oxidative stress in tissue culture. It is theorized that the protective effect due to cell density predominantly relates to intracellular factors such as reduced glutathione and extracellular factors such as catalase.

Spatiotemporal progression of cell death in the zone of ischemia surrounding burns.

Burns are dynamic injuries, characterized by progressive death of surrounding tissue over time. Although central to an understanding of burn injury progression, the spatiotemporal degrees and rates of cellular necrosis and apoptosis in the zone of ischemia surrounding burns are not well characterized. Using a validated porcine hot comb model, we probed periburn tissue at 1, 4, and 24 hours after injury for high-mobility group box 1 as a marker of necrosis and activated cleaved caspase-3 as a marker of apoptosis, followed by spatiotemporal morphometric analysis. We found that necrosis was the most prominent mechanism of cell death in burn injury progression, with significant progression between 1 and 4 hours postburn. Apoptosis appeared not to play a role in early burn injury progression but was observed in cells at the interface of necrotic and viable tissue at 24 hours postburn. Our findings imply that intervention within the first 4 hours following injury is likely necessary to limit burn injury progression. Additionally, based on high-mobility group box 1 staining patterns, we define distinct early, intermediate, and late pathological signs of cell necrosis that may facilitate delineation of causal mechanistic relationships of burn injury progression in vivo.

Validation of a vertical progression porcine burn model.

A major potential goal of burn therapy is to limit progression of partial- to full-thickness burns. To better test therapies, the authors developed and validated a vertical progression porcine burn model in which partial-thickness burns treated with an occlusive dressing convert to full-thickness burns that heal with scarring and wound contraction. Forty contact burns were created on the backs and flanks of two young swine using a 150 g aluminum bar preheated to 70°C, 80°C, or 90°C for 20 or 30 seconds. The necrotic epidermis was removed and the burns were covered with a polyurethane occlusive dressing. Burns were photographed at 1, 24, and 48 hours as well as at 7, 14, 21, and 28 days postinjury. Full-thickness biopsies were obtained at 1, 4, 24, and 48 hours as well as at 7 and 28 days. The primary outcomes were presence of deep contracted scars and wound area 28 days after injury. Secondary outcomes were depth of injury, reepithelialization, and depth of scars. Data were compared across burn conditions using analysis of variance and χ(2) tests. Eight replicate burns were created with the aluminum bar using the following temperature/contact-time combinations: 70/20, 70/30, 80/20, 80/30, and 90/20. The percentage of burns healing with contracted scars were 70/20, 0%; 70/30, 25%; 80/20, 50%; 80/30, 75%; and 90/20, 100% (P = .05). Wound areas at 28 days by injury conditions were 70/20, 8.1 cm(2); 70/30, 7.8 cm(2); 80/20, 6.6 cm(2); 80/30, 4.9 cm(2); and 90/20, 4.8 cm(2) (P = .007). Depth of injury judged by depth of endothelial damage for the 80/20 and 80/30 burns at 1 hour was 36% and 60% of the dermal thickness, respectively. The depth of injury to the endothelial cells 1 hour after injury was inversely correlated with the degree of scar area (Pearson's correlation r = -.71, P < .001). Exposure of porcine skin to an aluminum bar preheated to 80°C for 20 or 30 seconds results initially in a partial-thickness burn that when treated with an occlusive dressing progresses to a full-thickness injury and heals with significant scarring and wound contracture.

Fibronectin growth factor-binding domains are required for fibroblast survival.

Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg-Gly-Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10-100 nM. FN-null cells cultured on recombinant CCBD (FNIII(8-11)) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII(8-11) contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII(8-11) and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration.

Curcumin reduces injury progression in a rat comb burn model.

The oriental spice curcumin has anti-inflammatory and antioxidant effects. When given orally before injury, curcumin reduces burn progression in a rat comb burn model. The authors hypothesized that intravenous administration of curcumin after injury would reduce burn progression and that its effects are mediated through iron chelation. Two comb burns were created on the dorsum of Sprague-Dawley rats (weight, 300 g) using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds resulting in four rectangular 10 × 20 mm full-thickness burns separated by three 5 × 20 mm unburned interspaces (zone of ischemia). Animals were randomized to receive one of four doses of crude curcumin or one of six doses of purified curcumin intravenously 1 and 24 hours after injury. Another set of animals were randomized to deferoxamine or control vehicle. Wounds were observed at 7 days after injury for visual evidence of necrosis in the unburned interspaces. Full-thickness biopsies from the interspaces were evaluated with Hematoxylin and Eosin staining 7 days after injury for evidence of necrosis. The percentage of unburned interspaces undergoing necrosis at 1 week by purified curcumin doses was 0 µg/kg, 74%; 0.3 µg/kg, 58%; 1 µg/kg, 53%; 3 µg/kg, 37%; 10 µg/kg, 63%; 30 µg/kg, 53%; and 100 µg/kg, 26%. The differences among the groups were significant (P = .03). When compared with controls, the 1 and 3 µg/kg curcumin treatment groups had significantly less progression of interspaces to necrosis (P = .04 and .002) as did the 30 and 100 µg/kg treatment groups (P = .03 and <.001). Deferoxamine did not reduce burn progression. When administered intravenously 1 and 24 hours after injury, both crude and purified curcumin reduce the percentage of unburned interspaces that undergo necrosis in a rat hot comb burn model. The effects of purified curcumin appear to be bimodal, suggesting more than one mechanism of action. The effects of curcumin do not appear to be mediated by iron chelation.

Rosiglitazone, a PPAR-gamma ligand, reduces burn progression in rats.

Burns induce the activation of an inflammatory cascade that generates reactive oxygen radicals and lipid peroxidation leading to burn wound progression and extension. Peroxisome proliferation-activated receptor-gamma is a nuclear hormone receptor that is activated by transcription factors and plays an important role in the regulation of cellular proliferation and inflammation. We hypothesized that treatment of burns with rosiglitazone, a peroxisome proliferation-activated receptor-gamma ligand, would reduce burn wound progression. This is a randomized controlled study of 20 Sprague-Dawley rats. Two burns were created on each animal's dorsum using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds resulting in four rectangular 10 x 20 mm full thickness burns separated by three 5 x 20 mm unburned interspaces (zone of ischemia). Animals were randomized to rosiglitazone 4 mg/kg or vehicle by oral gavage 30 minutes after injury and at 24 and 48 hours after injury. Wounds were observed at 1, 2, 3, and 4 days after injury for visual evidence of necrosis in the unburned interspaces. Full thickness biopsies from the interspaces were evaluated with hematoxylin and eosin staining 7 days after injury for evidence of necrosis. The percentage of interspaces that progressed to necrosis was compared with chi tests. Forty comb burns with 120 unburned interspaces were evenly distributed between rosiglitazone and vehicle. The number of interspaces that progressed to full thickness necrosis at 1, 2, 3, 4, and 7 days after injury in the rosiglitazone and vehicle groups were 9/60 (15%) versus 13/60 (21%) (P = .48), 16/60 (27%) versus 15/60 (20%) (P = 1.00), 24/60 (40%) versus 46/60 (77%) (P = .001), 35/60 (58%) versus 53/60 (88%) (P = .001), and 43/60 (72%) versus 54/60 (90%) (P = .02), respectively. Treatment with oral rosiglitazone reduces the percentage of unburned skin interspaces that progress to full necrosis in a rat comb burn model.

Three-dimensional migration of human adult dermal fibroblasts from collagen lattices into fibrin/fibronectin gels requires syndecan-4 proteoglycan.

Fibroblast migration from the peri-wound collagenous stroma into the fibrin-laden wound is critical for granulation tissue formation and subsequent healing. Previously we found that fibroblast transmigration from a collagen matrix into a fibrin matrix required fibronectin (FN). Integrins alpha4beta1, alpha5beta1, and alphavbeta3 and dermatan sulfate CD44 were required for this invasive migration. Here we demonstrated that syndecan-4, a transmembrane heparan sulfate (HS) proteoglycan, known to bind FN, is also required for fibroblast invasive migration of a fibrin/FN gel. This conclusion was based on fibroblast migration using two independent means of disrupting syndecan-4: heparinase degradation of HS glycosaminoglycans or suppression of syndecan-4 core protein with antisense oligodeoxynucleotides. Isolated syndecan-4 from these fibroblasts bound Hep II recombinant constructs FN III12-V15>FN III12-15>FN III12-14 but did not bind the IIICS (V) domain. Furthermore, platelet-derived growth factor (PDGF), which is required to stimulate fibroblast migration, markedly increased cell levels of syndecan-4 core protein in a time and concentration-dependent fashion. PDGF also induced upregulation of syndecan-4 at transcriptional level as determined by RT-PCR. These results demonstrate that syndecan-4 is essential for fibroblast invasive migration into fibrin clot and that PDGF, the stimulus for migration, induces increased syndecan-4 core protein expression.

Fibroblast invasive migration into fibronectin/fibrin gels requires a previously uncharacterized dermatan sulfate-CD44 proteoglycan.

After tissue injury, fibroblast migration from the peri-wound collagenous stroma into the fibrin-laden wound is critical for granulation tissue formation and subsequent healing. Recently we found that fibroblast transmigration from a collagen matrix into a fibrin matrix required the presence of fibronectin. Several integrins-alpha 4 beta 1, alpha 5 beta 1, and alpha v beta 3-with known fibronectin binding affinity were necessary for this invasive migration. Here we examined another family of cell surface receptors: the proteoglycans. We found that dermatan sulfate was required for fibroblast migration into a fibronectin/fibrin gel. This conclusion was based on beta-xyloside inhibition of glycanation and specific glycosaminoglycan degradation. CD44, a cell surface receptor known to bind hyaluronan, not infrequently exists as a proteoglycan, decorated with various glycosaminoglycan chains including heparan sulfate and chondroitin sulfate, and as such can bind fibronectin. We found that CD44H, the non-spliced isoform of CD44, was necessary for fibroblast invasion into fibronectin/fibrin gels. Resting fibroblasts expressed mostly nonglycanated CD44H core protein, which became glycanated with chondroitin sulfate and dermatan sulfate, but not heparan sulfate, after a 24 h incubation with platelet-derived growth factor, the stimulus used in the migration assay. These results demonstrate that dermatan sulfate-CD44H proteoglycan is essential for fibroblast migration into fibrin clots and that platelet-derived growth factor, the stimulus for migration, induces the production of chondroitin-sulfate- and dermatan-sulfate-glycanated CD44H.