Jon E. Mogford

Effect of age and hypoxia on TGFβ1 receptor expression and signal transduction in human dermal fibroblasts: Impact on cell migration

Wound healing is critically affected by age, ischemia, and growth factors such as TGFβ1. The combined effect of these factors on fibroblast migration, an essential component of wound healing, is poorly understood. To address this deficiency, we examined expression of TGFβ receptor type I and II (TGFβRI and RII) under normoxia or hypoxia (1% O2) in cultured human dermal fibroblasts (HDFs) from young (ages 24–33) and aged (ages 61–73) adults. TGFβRI and RII expression was similar in both groups under normoxia. Hypoxia did not alter receptor levels in young HDFs but significantly decreased TGFβRI in aged cells (12 and 43%, respectively). Additionally, young cells displayed a 50% increase in activation of p42/p44 mitogen‐activated kinase by TGFβ1 (2–200 pg/ml) under hypoxia while aged cell levels of active p42/p44 decreased up to 24%. To determine functional outcomes of these findings, we measured the migratory capacity of the cells on type I collagen using a gold salt migration assay. Hypoxia increased the migratory index (MI) of young HDFs over normoxia by 30% but had no effect on aged cells. Under normoxia, TGFβ1 (1–1000 pg/ml) increased young HDF migration in a concentration‐dependent manner up to 109% over controls but minimally increased aged HDF migration (37%). Under hypoxia, TGFβ1 significantly increased young cell MI at all concentrations but was without effect on the aged HDF response. These data demonstrate that aged fibroblasts have an impaired migratory capacity with complete loss of responsiveness to hypoxia and deficits in the migratory and signal transduction responsiveness to TGFβ1 that may partly explain diminished healing capabilities often observed in aged patients. J. Cell. Physiol. 190: 259–265, 2002.

Wound epithelialization deficits in the transforming growth factor-α knockout mouse

In vitro, transforming growth factor‐α is an important factor controlling epithelial cell proliferation and migration. However, the transforming growth factor‐α knockout mouse has shown no wound epithelialization defect in tail amputation and full‐thickness back wounds. To resolve this disparity, we combined a full‐thickness head wound and a partial‐thickness ear wound on the transforming growth factor‐α knockout mouse for analysis of wound epithelialization with or without granulation tissue formation. Three‐millimeter ear wounds were made on the transforming growth factor‐α knockout and heterozygous control mice. Full‐thickness head wounds were made using a 6‐mm trephine on the crown of the skull. In the ear model, transforming growth factor‐α knockout mice had significantly larger epithelial gaps versus control at post‐operative day 3 and 5. Epithelial thickness at the wound edge of transforming growth factor‐α deficient mice was also depressed at post‐operative day 3 and post‐operative day 5 compared to control mice. On post‐operative day 8, most wounds of both groups were epithelialized. In contrast, no difference in epithelial gap or new granulation tissue was found in the head model. The data support the concept that transforming growth factor‐α plays a significant early role in wound epithelialization in vivo but its deficit is compensated if accompanied by granulation tissue formation. The data further show the importance of appropriate wound models to address the role of vulnerary factors.

Inhibition of prolyl 4-hydroxylase reduces scar hypertrophy in a rabbit model of cutaneous scarring

Hypertrophic scars result from excessive collagen deposition at sights of healing dermal wounds and can be functionally and cosmetically problematic. Pharmacological regulation of collagen synthesis and deposition is a direct approach to the control of scar tissue formation. We tested the ability of the phenanthrolinone derivative FG‐1648 (in 0.5% Carbopol 971 PNF gel, pH 6.5), a prolyl 4‐hydroxylase inhibitor, to reduce hypertrophic scar formation in a rabbit ear hypertrophic scar model. New Zealand White rabbits were divided into two treatment groups (n = 12 wounds per group with an equal number of controls): low‐dose group: 0.5% FG‐1648; high‐dose group: 1% FG‐1648. Left ears were used for treatment and right ear for control. Four 7‐mm dermal ulcer wounds were made on each ear. The inhibitor was topically applied to the wound at the time of wounding and once daily up to postoperative day 7. Wounds were harvested at postoperative day 28 and scar hypertrophy quantified by measurement of the scar elevation index. All wounds showed complete healing. Treatment of wounds with 1% prolyl 4‐hydroxylase inhibitor decreased the scar elevation index by 26% compared to control wounds (p < 0.01). Wounds treated with 0.5% FG‐1648 inhibitor showed no difference in scar elevation compared to control wounds. These results suggest that inhibition of prolyl 4‐hydroxylase may be a suitable agent for topical treatment for the prevention of hypertrophic scar tissue. (WOUND REP REG 2003;11:368–372)

A novel murine model of cyclical cutaneous ischemia-reperfusion injury

BACKGROUND Increasing evidence points to a principal role of ischemia-reperfusion in the pathogenesis of chronic skin ulceration, including pressure sores, diabetic ulcers, and venous ulcers. An incomplete understanding of this process and the limitations of current animal models of chronic wounds mandate a reproducible model in mice, in which transgenic and knockout technology are continually evolving. MATERIALS AND METHODS A murine model of chronic skin ulceration based on cyclical magnetic compression is presented. Forty-three C57BL/6J mice underwent varying degrees of cyclical compression with defined periods of reperfusion. Injury was measured grossly as regional necrosis, and tissue was harvested for histology, DNA electrophoresis, and reverse transcription polymerase chain reaction. RESULTS Skin necrosis became apparent only 12 h post cycling, and was cycle-responsive and quantitative in cycled subjects. Histopathologic analysis revealed a statistically significant doubling of the leukocyte count in sections from compressed skin versus sham controls. Moreover, apoptotic DNA laddering was evident in post ischemic skin and absent in controls. Real-time PCR analysis revealed a 300-fold higher expression in iNOS mRNA from cyclically compressed skin compared with normal skin: such expression was temporal in nature. CONCLUSIONS A murine model of pressure necrosis, which bears all of the gross, histological, and molecular features of ischemia-reperfusion injury, has been established. Application of this model to the vast number of transgenic mice available will further our understanding of the mechanism of pressure sore development.

Fibrin sealant combined with fibroblasts and platelet‐derived growth factor enhance wound healing in excisional wounds

We test the hypothesis that the fibrinogen‐thrombin formulation of fibrin sealant combined with fibroblasts and PDGF‐BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full‐thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 × 105 cells/wound) embedded in the fibrinogen component. At post‐wounding day 7, there was no difference in the diluted vs. non‐diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF‐BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two‐fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant.

Inhibition of procollagen C-proteinase reduces scar hypertrophy in a rabbit model of cutaneous scarring

Hypertrophic scarring, which results from excessive collagen deposition at sites of dermal wound repair, can be functionally and cosmetically debilitating to the surgical patient. Pharmacological regulation of collagen synthesis and deposition is a direct approach to the control of scar tissue formation. One of the key steps in collagen stabilization is the cleavage of the C‐terminal propeptide from the precursor molecule to form collagen fibrils, a reaction catalyzed by procollagen C‐proteinase (PCP). We tested the ability of a PCP inhibitor to reduce hypertrophic scar formation in a rabbit ear model. After the placement of four, 7‐mm dermal wounds on each ear, New Zealand white rabbits received PCP inhibitor subcutaneously in the left ear at four time points postwounding: days 7, 9, 11, 13 (early treatment; n=20 wounds) or days 11, 13, 15, 17 (late treatment; n=20 wounds). The right ear of each animal served as a control (vehicle alone). Wounds were harvested on postoperative day 28 and scar hypertrophy quantified by measurement of the scar elevation index. Early treatment of wounds with PCP inhibitor did not reduce scar formation compared with controls (p>0.05). However, late treatment resulted in a statistically significant reduction in the scar elevation index (p<0.01). Our results point not only to the potential use of PCP inhibitors to mitigate hypertrophic scarring but also to the temporal importance of drug delivery for antiscarring therapy.

Silicone Occlusive Treatment of Hypertrophic Scar in the Rabbit Model

BACKGROUND Hypertrophic scar formation at sites of healed cutaneous injury often produces functional and esthetic deficits. Treatments have been limited in part by a lack of understanding of scar etiology and the lack of animal models of hypertrophic scarring. Silicone dressing is reported to provide positive outcomes with respect to a reduction in scar hypertrophy and an improvement in color differences, although the exact mechanism is unknown. OBJECTIVE We tested the effectiveness of silicone adhesive gel in the reduction of scar hypertrophy in an animal model of scarring. METHODS Silicone adhesive gel was applied to scars in a rabbit ear model of hypertrophic scarring. Scarring in this model, which displays reduced hypertrophy in response to steroid injections and aging similar to that of human beings, was measured by the Scar Elevation Index (SEI), a ratio of the scar height over normal skin, in which readings greater than 1.0 represent a raised scar. RESULTS SEIs were significantly reduced after 4-week applications of silicone gel (1.15 +/- 0.15 vs 1.71 +/- 0.33, respectively; P < .001) versus untreated scars. Nonsilicone control dressings did not alter SEIs in comparison with those found for controls. No histologic differences in scar cellularity, inflammation, or matrix organization were found between treatment groups; however, ultrastructural observation revealed numerous vacuoles in basal cells of control and nonsilicone-treated scars that were not found in unwounded skin or silicone gel-treated scars. The similarity in water vapor transmission rates for silicone gel and a nonsilicone dressing eliminated scar hydration as the sole mechanism of action of the silicone dressings. CONCLUSIONS Our findings with the rabbit model demonstrate the effectiveness of silicone gel for hypertrophic scar treatment and confirm the usefulness of this model for further study of the mechanism of occlusion. (Aesthetic Surg J 2002;22:147-153.).

Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence

A central question in cell biology is how cells become senescent. After a finite number of cell divisions, normal cultured human cells enter a state of irreversible growth arrest, termed “replicative senescence.” Alternatively, oxidative stress in the form of hydrogen peroxide (H2O2) can render human dermal fibroblasts (HDFs) nonproliferative and quiescent, a phenomenon known as stress‐induced premature senescence (SIPS). Although critical to the understanding of the pathophysiological basis of many diseases, there is no research to date that has simultaneously examined the interactions between age, oxidative stress, and SIPS. Therefore, the goals of this study were to examine in concert the interactions between these three factors in primary HDFs, and to test our central hypothesis that aging lowers the ability of primary HDFs to respond to oxidative stress. Our data provide, for the first time, evidence that aging dramatically reduces the capacity of primary HDFs to respond to the challenge of hydrogen peroxide. Specifically, aged HDFs showed decreased cell viability, decreased phosphorylation (activation) of pro‐survival kinases (Akt and ERK 1/2), and increased entrance into a senescent state when compared with their younger counterparts. Another important conclusion of this study is that blockade of transforming growth factor‐β1 had a pronounced “rescue effect” in the aged, preventing entrance of HDFs into cellular senescence.

Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing.

BACKGROUND Tissue ischemia and aging are independent features associated with the healing impairment of cutaneous wounds. However, the pathophysiology of these processes as they relate to impaired-healing wounds is poorly understood. MATERIALS AND METHODS A single full-thickness biopsy wound was made on both ears of young (3-6 month) and aged (>24 month) Fisher rats. One ear was rendered ischemic by transection of the vasculature at the ear base, while the other ear served as an internal nonischemic control. Wounds were harvested from 3 to 7 days and were evaluated histologically for either granulation tissue formation and epithelialization. Total RNA from wounds harvested at postoperative day 7 was probed using a nylon-based cDNA array to assess global genetic expression alterations. RESULTS Healing in the rat ear model is impaired by both ischemia and advanced age as measured by granulation tissue formation and wound epithelialization. Granulation tissue formation was affected to a greater degree by ischemia than age (-58% versus -21%, respectively) while epithelialization displayed an opposite response (-17% versus -53%, respectively). Global analysis of gene expression suggests that ischemia engenders a marked increase in genes displaying altered expression in aged animals compared to young animals. Importantly, all possible alterations in gene expression are found in samples from aged ischemic wounds, indicating that gene regulation is not simply depressed by advanced age. CONCLUSIONS Wound epithelialization appears to be affected to a greater degree by advanced age than by ischemia. The results demonstrate the distinctive phenotype presented by the clinically relevant combination of age and ischemia in an in vivo model of cutaneous wound healing.

Delayed wound healing in Mac-1-deficient mice is associated with normal monocyte recruitment.

The Mac‐1 integrin is an important mediator of migration and inflammatory activation of neutrophils and monocytes. However, the role of Mac‐1 in modulating macrophage emigration and activation and its subsequent impact on cutaneous wound healing have not been fully elucidated. To examine the significance of Mac‐1 to murine wound healing, we measured epithelialization and granulation tissue formation in partial‐thickness ear wounds and full‐thickness head wounds, respectively, in Mac‐1–deficient mice. Wounds were histologically analyzed at postwounding days 3, 5, and 7. The gap measured between the leading edges of inward‐migrating granulation tissue was significantly increased in knockout mice compared with control animals at day 5 (3.8±0.3 vs. 2.6±0.5 mm; p<0.001) and day 7 (2.2±0.4 vs. 0.96±0.73 mm; p=0.005). Epithelial gap measurements were also increased in knockout mice vs. wild‐type controls at days 3 (0.62±0.02 vs. 0.54±0.07 mm; p<0.05) and 5 (0.58±0.06 vs. 0.39±0.08 mm; p<0.001). Immunohistochemistry showed equal numbers of macrophages in knockout and control wounds. These findings show that Mac‐1 is required for normal wound healing but that the attenuation in the deposition of granulation tissue and wound epithelialization in Mac‐1 knockout mice is not associated with decreased monocyte migration into the wound.