Judith A. Mack

Hoxb13 knockout adult skin exhibits high levels of hyaluronan and enhanced wound healing

In contrast to adult cutaneous wound repair, early gestational fetal cutaneous wounds heal by a process of regeneration, resulting in little or no scarring. Previous studies indicate that down‐regulation of HoxB13, a member of the highly conserved family of Hox transcription factors, occurs during fetal scarless wound healing. No down‐regulation was noted in adult wounds. Here, we evaluate healing of adult cutaneous wounds in Hoxb13 knockout (KO) mice, hypothesizing that loss of Hoxb13 in adult skin should result in enhanced wound healing. Tensiometry was used to measure the tensile strength of incisional wounds over a 60‐day time course; overall, Hoxb13 KO wounds are significantly stronger than wild‐type (WT). Histological evaluation of incisional wounds shows that 7‐day‐old Hoxb13 KO wounds are significantly smaller and that 60‐day‐old Hoxb13 KO wounds exhibit a more normal collagen architecture compared with WT wounds. We also find that excisional wounds close at a faster rate in Hoxb13 KO mice. Biochemical and histochemcial analyses show that Hoxb13 KO skin contains significantly elevated levels of hyaluronan. Because higher levels of hyaluronan and enhanced wound healing are characteristics of fetal skin, we conclude that loss of Hoxb13 produces a more “fetal‐like” state in adult skin.

Enhanced Inflammation and Accelerated Wound Closure Following Tetraphorbol Ester Application or Full-Thickness Wounding in Mice Lacking Hyaluronan Synthases Has1 and Has3

Hyaluronan (HA) is an abundant matrix molecule whose functions in the skin remain to be fully defined. To explore the roles of HA in cutaneous injury responses, double-knockout mice (abbreviated as Has1/3 null) that lack two HA synthase enzymes (Has1 and Has3) but still express functional Has2, were used in two types of experiments: (i) application of 12-O-tetradecanoylphorbol-13-acetate (TPA), and (ii) full-thickness wounding of the skin. Uninjured Has1/3 null mice were phenotypically normal. However, after TPA, the accumulation of HA that normally occurs in wildtype epidermis was blunted in Has1/3 null epidermis. In excisional wound healing experiments, wound closure was significantly faster in Has1/3 null than in wildtype mice. Coincident with this abnormal wound healing, a marked decrease in epidermal and dermal HA and a marked increase in neutrophil efflux from cutaneous blood vessels were observed in Has1/3 null skin relative to wildtype skin. Has1/3 null wounds displayed an earlier onset of myofibroblast differentiation. In summary, selective loss of Has1 and Has3 leads to a pro-inflammatory milieu that favors recruitment of neutrophils and other inflammation-related changes in the dermis.

Wounding-Induced Synthesis of Hyaluronic Acid in Organotypic Epidermal Cultures Requires the Release of Heparin-Binding EGF and Activation of the EGFR

Hyaluronic acid (HA), a glycosaminoglycan located between keratinocytes in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat keratinocyte organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to 24 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by 24 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive factor. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble factor. From earlier evidence, we hypothesized that an EGF-like growth factor might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (2) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like growth factor (HB-EGF) blocked wounding-induced HA synthesis by > or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.

Roles of Proteoglycans and Glycosaminoglycans in Wound Healing and Fibrosis

A wound is a type of injury that damages living tissues. In this review, we will be referring mainly to healing responses in the organs including skin and the lungs. Fibrosis is a process of dysregulated extracellular matrix (ECM) production that leads to a dense and functionally abnormal connective tissue compartment (dermis). In tissues such as the skin, the repair of the dermis after wounding requires not only the fibroblasts that produce the ECM molecules, but also the overlying epithelial layer (keratinocytes), the endothelial cells, and smooth muscle cells of the blood vessel and white blood cells such as neutrophils and macrophages, which together orchestrate the cytokine-mediated signaling and paracrine interactions that are required to regulate the proper extent and timing of the repair process. This review will focus on the importance of extracellular molecules in the microenvironment, primarily the proteoglycans and glycosaminoglycan hyaluronan, and their roles in wound healing. First, we will briefly summarize the physiological, cellular, and biochemical elements of wound healing, including the importance of cytokine cross-talk between cell types. Second, we will discuss the role of proteoglycans and hyaluronan in regulating these processes. Finally, approaches that utilize these concepts as potential therapies for fibrosis are discussed.

Altered serotonin immunoreactivities in the left colon of patients with colonic inertia

Serotonin is an important positive regulator of colonic motility and transit. Its quantity and distribution in the left colon could be abnormal in patients with colonic inertia (CI) and contribute to the disease.

Hyperglycemia-Induced Changes in Hyaluronan Contribute to Impaired Skin Wound Healing in Diabetes: Review and Perspective.

Ulcers and chronic wounds are a particularly common problem in diabetics and are associated with hyperglycemia. In this targeted review, we summarize evidence suggesting that defective wound healing in diabetics is causally linked, at least in part, to hyperglycemia-induced changes in the status of hyaluronan (HA) that resides in the pericellular coat (glycocalyx) of endothelial cells of small cutaneous blood vessels. Potential mechanisms through which exposure to high glucose levels causes a loss of the glycocalyx on the endothelium and accelerates the recruitment of leukocytes, creating a proinflammatory environment, are discussed in detail. Hyperglycemia also affects other cells in the immediate perivascular area, including pericytes and smooth muscle cells, through exposure to increased cytokine levels and through glucose elevations in the interstitial fluid. Possible roles of newly recognized, cross-linked forms of HA, and interactions of a major HA receptor (CD44) with cytokine/growth factor receptors during hyperglycemia, are also discussed.

Coincidence of Connective Tissue Growth Factor Expression with Fibrosis and Angiogenesis in Postoperative Peritoneal Adhesion Formation

Purpose: To investigate the relationship between connective tissue growth factor (CTGF) and fibrosis and angiogenesis in postoperative peritoneal adhesion formation. Methods: Adhesions were performed in 35 rats by creation of a peritoneal patch. Animals were sacrificed at 7 different time-points over 3 weeks. Adhesions and uninjured peritoneum from all animals were assessed by Northern blotting for CTGF and collagen-I mRNA and by immunohistochemistry for CTGF localization, degree of fibrosis and angiogenesis. Results: Persistent adhesions formed in all animals. CTGF and collagen-I mRNA were increased in adhesions compared to uninjured peritoneum (p < 0.05 for both). The temporal expression pattern depicted delayed peak levels of collagen-I mRNA with increasing tendency for both transcripts at the end of the observation period. Fibrosis within adhesions correlated positively with time after surgery (r = 0.85; p < 0.001) and showed typical signs of chronic tissue fibrosis at later time points. Angiogenesis was detected in adhesions but not in uninjured peritoneum (p = 0.001) and coincided with the spatial and temporal expression of CTGF protein in fibroblasts and vascular endothelial cells. Conclusions: The co-expression of CTGF with increasing fibrosis and angiogenesis in postoperative peritoneal adhesions suggests a role for CTGF as critical molecule in fibrous adhesive disease and target for future adhesion prevention.

Reduced expression of serotonin receptor(s) in the left colon of patients with colonic inertia

AbstractPURPOSE: Serotonin regulates colonic motility via receptors expressed on neural fibers and smooth muscle. Colonic inertia is characterized by delayed colonic transit. Abnormalities in serotonin receptor protein, as judged by immunoreactivity levels, could contribute to the origin of colonic inertia. The aim of this study was to investigate the expression of serotonin receptor(s) immunoreactivity in the left colon of patients with colonic inertia compared with controls. METHODS: Sixteen patients who underwent subtotal colectomy for colonic inertia were assessed. Colonic transit time was measured with the radiopaque marker technique and presented as the number of retained markers in the colon on Day 5. The control group consisted of 18 patients who underwent left hemicolectomy for colonic carcinoma; histologically normal tissues from the left colon were used. Immunohistochemical staining for serotonin receptor was performed with a rabbit anti-idiotypic antibody. The average positive area (square pixels) in the mucosa, muscularis mucosa, submucosa, and circular and longitudinal muscles per microscopic field (63×) was calculated based on measurement of the positively stained area in 20 randomly chosen microscopic fields in each related structure. The Scion Image computer analysis system was used. RESULTS: Serotonin receptor(s) immunoreactivity was mainly detected in the muscular mucosa, circular muscles, and longitudinal muscles and rarely in the mucosa and submucosa. In muscularis mucosa and circular muscle, the positive areas were significantly less in the colonic inertia group than in controls (muscularis mucosa: 29.1 ± 10.8 vs. 109.7 ± 28.2, P < 0.05; circular muscle: 25.6 ± 6.2 vs.90.2 ± 19.1, P < 0.01). There were significantly positive correlations in the control group in serotonin receptor(s) immunoreactivity levels between circular muscle and longitudinal muscle (r = 0.54, P < 0.05) and between muscular mucosa and longitudinal muscle (r = 0.57, P < 0.05) but not in colonic inertia patients. In addition, the positive areas in the circular muscle were positively correlated to the colonic transit time (Spearman’s rank correlation, 0.83; P < 0.01). CONCLUSION: In colonic inertia patients, the serotonin receptor(s) immunoreactivity level is lower in muscular mucosa and circular muscle. The absence of a correlation of serotonin receptor(s) immunoreactivity in the muscular mucosa and muscularis propria in the patient group implies that an uncoordinated expression of serotonin receptors may also contribute to colonic inertia. However, the positive correlation between serotonin receptor(s) immunoreactivity levels in the circular muscle and the transit time observed in colonic inertia patients suggests a decrease in stimulatory subtypes and at the same time an increase in inhibitory subtypes of serotonin receptors in this tissue.

Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress

Background: Hyaluronan (HA), an extracellular glycosaminoglycan, is normally produced by three HA synthase (Has) enzymes. Results: Skin fibroblasts from Has1/Has3 double knock-out mice have higher Has2 expression and HA levels and are resistant to cell death after UVB exposure or serum starvation. Conclusion: HA modulates injury-induced apoptotic responses in fibroblasts. Significance: HA has an important role in cell death responses. A balanced turnover of dermal fibroblasts is crucial for structural integrity and normal function of the skin. During recovery from environmental injury (such as UV exposure and physical wounding), apoptosis is an important mechanism regulating fibroblast turnover. We are interested in the role that hyaluronan (HA), an extracellular matrix molecule synthesized by HA synthase enzymes (Has), plays in regulating apoptosis in fibroblasts. We previously reported that Has1 and Has3 double knock-out (Has1/3 null) mice show accelerated wound closure and increased numbers of fibroblasts in the dermis. In the present study, we report that HA levels and Has2 mRNA expression are higher in cultured Has1/3 null primary skin fibroblasts than in wild type (WT) cells. Apoptosis induced by two different environmental stressors, UV exposure and serum starvation (SS), was reduced in the Has1/3 null cells. Hyaluronidase, added to cultures to remove extracellular HA, surprisingly had no effect upon apoptotic susceptibility to UVB or SS. However, cells treated with 4-methylumbelliferone to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. When fibroblasts were transfected with Has2-specific siRNA that lowered Has2 mRNA and HA levels by 90%, both Has1/3 null and WT cells became significantly more sensitive to apoptosis. The exogenous addition of high molecular weight HA failed to reverse this effect. We conclude that Has1/3 null skin fibroblasts (which have higher levels of Has2 gene expression) are resistant to stress-induced apoptosis.

Persistent Inflammation and Angiogenesis during Wound Healing in K14-Directed Hoxb13 Transgenic Mice

Chronic, nonhealing wounds and inadequate tissue repair characterized by excessive fibrosis continue to have a considerable negative effect on health and quality of life. Understanding the molecular events required for adequate healing, including the transcriptional control of wound repair, will be important for the development of future therapies. We previously showed that loss of Hoxb13 from murine skin results in enhanced cutaneous wound healing, suggesting that Hoxb13 has a negative effect on wound repair. To test this, we generated skin-specific Hoxb13 transgenic (TG) mice that overexpress Hoxb13 in the basal layer of the epidermis by the human keratin 14 promoter. Using these mice, we evaluated the effects of Hoxb13 overexpression on cutaneous wound healing. Transgenic wounds were characterized by persistence of the fibrin clot and prolonged inflammation. Notably, neutrophils, which had cleared from wild-type wounds, were still pronounced in TG wounds. Marked epidermal hyperplasia was observed at TG wound edges, and dermal vessels were grossly abnormal compared with wild-type mice. Both vascular endothelial growth factor and tumor necrosis factor-alpha were upregulated in Hoxb13 TG skin. Together, our results identify Hoxb13 as a potential important clinical target in wound healing and other pathologies characterized by abnormal or excessive inflammation, angiogenesis, or epidermal proliferation.