Nancy L. Cardwell

The tumorigenic and angiogenic effects of MGSA/GRO proteins in melanoma.

Continuous expression of the MGSA/GROα, β, or γ chemokine bestows tumor‐forming capacity to the immortalized murine melanocyte cell line, melan‐a. The mechanism for this transformation is unclear, although both autocrine and paracrine processes are possible because melan‐a cells as well as endothelial cells express a low level of the receptor for this ligand. To further define the role of MGSA/GRO proteins in melanocyte transformation, two types of experiments were designed to neutralize the biological effects of MGSA/GRO in the transfected melan‐a clones: (1) the effect of neutralizing antiserum to MGSA/GRO proteins on melan‐a tumor growth was assessed; (2) the tumor‐forming capacity of melan‐a clones expressing ELR motif‐mutated forms of MGSA/GRO with compromised receptor affinity was compared to the tumor‐forming capacity of clones expressing wild‐type MGSA/GRO. These experiments revealed that SCID mice inoculated with MGSA/GROα‐ or γ‐expressing melan‐a cells and subsequently treated with antiserum to the respective chemokine exhibited decreased tumor growth. This reduction in tumor growth was accompanied by declining angiogenic activity in MGSA/GROγ‐expressing tumors. Moreover, athymic nude mice injected with melan‐a cells expressing ELR‐mutant forms of MGSA/GROα exhibited markedly impaired tumor‐forming capacity compared with those mice injected with melan‐a clones expressing wild‐type MGSA/GRO. These data suggest that continuous expression of MGSA/GRO proteins may facilitate tumor growth by stimulating the growth of microvessels into the tumor (paracrine) and by affecting melanocyte growth (autocrine). J. Leukoc. Biol. 67: 53–62; 2000.

Calreticulin Enhances Porcine Wound Repair by Diverse Biological Effects

Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds.

Boosting epidermal growth factor receptor expression by gene gun transfection stimulates epidermal growth in vivo

Expression constructs encoding a full‐length cDNA encoding the human epidermal growth factor receptor, or reporter gene for green fluorescent protein or luciferase were coated onto gold particles and driven into porcine skin using a gene gun delivery system. Strategies for epidermal growth factor receptor boosting were tested in two types of wounds. For grafted wounds, intact porcine skin was pretreated by the introduction of the epidermal growth factor receptor expression construct 24 hours before its harvesting as a split‐thickness skin graft. Partial‐thickness excisional wound beds (donor sites) were transfected at the time of their creation. Wound healing parameters were subsequently tested in the presence or absence of excess epidermal growth factor ligand. Initial distributions of gene gun delivered gold particles as well as luciferase expression levels suggested that optimal skin penetrations and expression levels were achieved at 500 psi for intact epidermis and 300 psi for exposed wound beds. At 2 days after gene delivery, visualization of green fluorescent protein by fluorescence microscopy showed focal expression of green fluorescent protein at the advancing epithelial outgrowths found at wound edges or surviving epithelial remnants. Green fluorescent protein expression appeared transient since no green fluorescent protein was noted in specimens removed at 4 days after injury. Northern blot analysis on mRNA isolated from wounds 2 days after introduction of epidermal growth factor receptor coated gold particles by gene gun confirmed the expression of the human epidermal growth factor receptor transgene in both skin grafts and excisional wounds. Skin grafts showed subsequent biological responses to the introduction of excessive epidermal growth factor receptor as well as expression of the human epidermal growth factor receptor construct within healing epidermis. While control autografts (reporter gene treated, epidermal growth factor alone, placebo formula, no treatment) showed few 5′‐bromodeoxyuridine‐labeled cells, epidermal growth factor receptor autografts showed 5′‐bromodeoxyuridine labeling of nearly every basal cell. Favorable wound healing outcomes were also shown within excisional wounds following in vivo boosting of epidermal growth factor receptor. Four days after receiving epidermal growth factor receptor particle bombardment, resurfacing was significantly accelerated in those wounds receiving epidermal growth factor receptor transgene. Application of topical epidermal growth factor ligand resulted in the highest percentage of resurfacing. Maximal re‐epithelialization was noted in wound beds receiving both receptor boosting and excessive daily epidermal growth factor ligand. A modest increase in the thickness of the granulation tissue followed gene therapy with epidermal growth factor receptor. In summary these in vivo data suggest that it is possible to boost in vivo expression of a tyrosine kinase receptor during wound repair. Increased epidermal growth factor receptor expression has an integral impact on cell proliferation, rates of resurfacing and dermal components and merits consideration as a possible therapeutic agent.

The effectiveness of sodium 2-mercaptoethane sulfonate (mesna) in reducing capsular formation around implants in a rabbit model.

The development of capsular contracture is the most common complication associated with the insertion of breast implants. The authors studied the role of sodium 2-mercaptoethane sulfonate (mesna) in reduction of capsular formation in a rabbit model. Two 40-cc textured saline implants were placed dorsally into each of the 20 rabbits in the study. At the time of insertion of the implants, 10 ml of a 10% solution of mesna was instilled into one of the pockets and normal saline was instilled into the other. The implants were removed and a capsulectomy was performed at 5 months. The capsules were examined histologically for qualitative differences between the two groups. Quantitative analysis of the thickness of the capsule and the myofibroblast populations was also performed and compared between the two groups. The mean total thickness of the capsule around the implants was 496.8 &mgr;m in the mesna-treated group compared with 973.7 &mgr;m in the saline-treated group (p < 0.001). Likewise, the thickness of the myofibroblast layer was reduced in the mesna-treated group at 283.2 &mgr;m versus 555 &mgr;m in the saline-treated group (p < 0.0001). The capsules were also relatively less vascular in the mesna-treated group. Because of its ability to reduce the extent of capsular formation and to diminish development of myofibro-blasts in the capsules, mesna would appear to be a useful adjunct in the prevention of capsular contracture formation.

Hydrophilic polymers enhance early functional outcomes after nerve autografting.

BACKGROUND Approximately 12% of operations for traumatic neuropathy are for patients with segmental nerve loss, and less than 50% of these injuries obtain meaningful functional recovery. Polyethylene glycol (PEG) therapy has been shown to improve functional outcomes after nerve severance, and we hypothesized this therapy could also benefit nerve autografting. METHODS We used a segmental rat sciatic nerve injury model in which we repaired a 0.5-cm defect with an autograft using microsurgery. We treated experimental animals with solutions containing methylene blue (MB) and PEG; control animals did not receive PEG. We recorded compound action potentials (CAPs) before nerve transection, after solution therapy, and at 72 h postoperatively. The animals underwent behavioral testing at 24 and 72 h postoperatively. After we euthanized the animals, we fixed the nerves, sectioned and immunostained them to allow for quantitative morphometric analysis. RESULTS The introduction of hydrophilic polymers greatly improved morphological and functional recovery of rat sciatic axons at 1-3 d after nerve autografting. Polyethylene glycol therapy restored CAPs in all animals, and CAPs were still present 72 h postoperatively. No CAPS were detectable in control animals. Foot Fault asymmetry scores and sciatic functional index scores were significantly improved for PEG therapy group at all time points (P < 0.05 and P < 0.001; P < 0.001 and P < 0.01). Sensory and motor axon counts were increased distally in nerves treated with PEG compared with control (P = 0.019 and P = 0.003). CONCLUSIONS Polyethylene glycol therapy improves early physiologic function, behavioral outcomes, and distal axonal density after nerve autografting.

Noncontact imaging of burn depth and extent in a porcine model using spatial frequency domain imaging

The standard of care for clinical assessment of burn severity and extent lacks a quantitative measurement. In this work, spatial frequency domain imaging (SFDI) was used to measure 48 thermal burns of graded severity (superficial partial, deep partial, and full thickness) in a porcine model. Functional (total hemoglobin and tissue oxygen saturation) and structural parameters (tissue scattering) derived from the SFDI measurements were monitored over 72 h for each burn type and compared to gold standard histological measurements of burn depth. Tissue oxygen saturation (stO₂) and total hemoglobin (ctHbT) differentiated superficial partial thickness burns from more severe burn types after 2 and 72 h, respectively (p < 0.01), but were unable to differentiate deep partial from full thickness wounds in the first 72 h. Tissue scattering parameters separated superficial burns from all burn types immediately after injury (p < 0.01), and separated all three burn types from each other after 24 h (p < 0.01). Tissue scattering parameters also showed a strong negative correlation to histological burn depth as measured by vimentin immunostain (r² > 0.89). These results show promise for the use of SFDI-derived tissue scattering as a correlation to burn depth and the potential to assess burn depth via a combination of SFDI functional and structural parameters.

CM101 stimulates cutaneous wound healing through an anti-angiogenic mechanism

CM101, an anti-pathoangiogenic polysaccharide derived from group B streptococcus, has been shown to inhibit inflammatory angiogenesis and accelerate wound healing in a mouse model and minimize scarring/gliosis following spinal cord injury. To evaluate the in vivo effects of CM101 on cutaneous wound healing in the pig, intravenously delivered CM101 or placebo vehicle was given 1 h after cutaneous wounding and again at 72 h after injury. Tissues from partial-thickness and full-thickness excisions were collected at days 4 and 7 after wounding and evaluated for a variety of standard healing parameters. Both types of CM101-treated wounds showed significantly less evidence of inflammatory angiogenesis when assessed by macroscopic photography of the wound surface, qualitative histological observations, laser doppler perfusion imaging, and quantitative morphometric analysis of microvessel area from endothelium selectively immunostained for factor VIII. Resurfacing was accelerated in partial-thickness and full-thickness excisions that received two doses of CM101 as compared to the placebo-treated excisional wounds. Neodermal thickness was increased in CM101-treated wounds at day 4 and was slightly reduced in comparison with placebo by day 7. New collagen accumulation appeared to be unaffected by the CM101 treatment. Immunohistochemical staining using a polyclonal antisera directed against the anti-pathoangiogenic CM101 target protein HP59 on day 7 indicated a strong immunoreactivity on the microvessels present in the control wounds but not in wounds of the CM101-treated animals. In summary, the immunolocalization HP59 in the microvessels of the cutaneous wound bed in control but not in CM101 treated wounds suggests that CM101 inhibits the pathologic inflammatory angiogenesis accompanying the normal granulation processes. The net biological effect of inhibited inflammatory pathoangiogenesis is a diminished, suggested and purely physiologic, microvascular bed which translates into an enhanced rate of epithelial resurfacing and therefore an overall accelerated rate of wound repair.

Dual-imaging system for burn depth diagnosis

Currently, determination of burn depth and healing outcomes has been limited to subjective assessment or a single modality, e.g., laser Doppler imaging. Such measures have proven less than ideal. Recent developments in other non-contact technologies such as optical coherence tomography (OCT) and pulse speckle imaging (PSI) offer the promise that an intelligent fusion of information across these modalities can improve visualization of burn regions thereby increasing the sensitivity of the diagnosis. In this work, we combined OCT and PSI images to classify the degree of burn (superficial, partial-thickness and full-thickness burns). Algorithms were developed to integrate and visualize skin structure (with and without burns) from the two modalities. We have completed the proposed initiatives by employing a porcine burn model and compiled results that attest to the utility of our proposed dual-modal fusion approach. Computer-derived data indicating the varying burn depths were validated through immunohistochemical analysis performed on burned skin tissue. The combined performance of OCT and PSI modalities provided an overall ROC-AUC=0.87 (significant at p<0.001) in classifying different burn types measured after 1-h of creating the burn wounds. Porcine model studies to assess feasibility of this dual-imaging system for wound tracking are underway.

RAG2-/-, I kappa B-alpha-/- chimeras display a psoriasiform skin disease.

Nuclear factor-κB, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-κB activity is regulated by inhibitor of κB (IκB), the gene encoding IκB-α was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-κB. IκB-α–/– mice have constitutive nuclear factor-κB activity, severe skin disease, and neonatal lethality. To determine the role of IκB-α deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2–/–, IκB-α–/– chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2–/– recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-κB, due to deficiency in IκB-α, can invoke severe psoriasiform dermatitis in adult mice.

RAG2–/–, IκB-α–/– Chimeras Display a Psoriasiform Skin Disease

Nuclear factor-κB, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-κB activity is regulated by inhibitor of κB (IκB), the gene encoding IκB-α was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-κB. IκB-α–/– mice have constitutive nuclear factor-κB activity, severe skin disease, and neonatal lethality. To determine the role of IκB-α deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2–/–, IκB-α–/– chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2–/– recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-κB, due to deficiency in IκB-α, can invoke severe psoriasiform dermatitis in adult mice.