Histochemical and Morphometrical Analyses of Scarless Wound Healing in Mouse Fetal Model.

OBJECTIVE: Scar formation is an inevitable outcome after craniofacial surgery in the congenital facial anomaly. Scarless healing is the ultimate treatment after the surgery. Therefore, we elucidate the mechanism underlying scarless healing during fetal development. METHODS: A full-thickness back skin excision (1 × 0.5 mm) was made at embryonic day 16.5 (E16.5) and 18.5 (E18.5) in fetal C57BL/6J mice and examined the histochemical and morphometrical findings of wound healing after 48 hours. RESULTS: The wound made at E16.5 showed almost complete re-epithelialization with fine reticular dermal collagen fibers, but not at E18.5. The ratio of CK5 positive area was significantly higher in the wound of E16.5 operation than in the E18.5. The wounds made at E18.5 showed granulation tissue formation which will lead to subsequent scar formation. The collagen fibers tended to be thinner in wound than in normal skin, while the decrease in the number of fibers but the increase in the straightness of fibers were evident in the wound at E18.5. CONCLUSION: Transition point of scarless healing seemed between E16.5 and E18.5 in mice, which may imply that the potential of epithelial regeneration and matrix formation was changed, possibly due to alteration of cell constitution and decrease in stemness, at that time.

MK5 haplodeficiency decreases collagen deposition and scar size during post-myocardial infarction wound repair.

MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/- and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/- mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/- mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/- hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/- hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/- hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/- hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/- mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5-/- mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.

Effect of dual mTOR inhibitor on TGFß1-induced fibrosis in primary human urethral scar fibroblasts.

BACKGROUND: TGFß1 and mTOR are considered to play important roles in fibrotic diseases. Rapamycin has been reported to inhibit urethral stricture formation in a rabbit model of urethral fibrosis. AIM: To evaluate if dual mTOR inhibitor has a superior efficacy compared with rapamycin on inhibiting cell proliferation and collagen expression in human urethral scar fibroblasts (HUSFs). METHODS: We established HUSF cultures from fresh surgical specimen. The HUSFs were identified with typical fibroblast markers using immunofluorescence. Then we examined the effect of TGFß1 on HUSFs using Cell Counting Kit-8 and Western blot. The inhibiting effects of OSI-027 (a dual mTOR inhibitor) on cell proliferation and collagen expression in TGFß1-induced HUSFs were compared with rapamycin using Cell Counting Kit-8, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: HUSFs were stained positive for vimentin, collagen I, and collagen III. TGFß1 had no effect on cell proliferation but increased collagen I and collagen III expressions in HUSFs. OSI-027 was more effective inhibiting cell proliferation and collagen expression compared with rapamycin in TGFß1-induced HUSFs. OSI-027 played a more important role in inhibiting TGFß1-induced mTOR pathway and phosphorylation of Smad2 compared with rapamycin in HUSFs. CONCLUSION: OSI-027 can inhibit the pro-fibrotic effects of TGFß1 significantly compared with rapamycin in HUSFs. These findings may provide a new therapy in the adjunctive treatment of urethral stricture disease.

The proto-oncogene tyrosine protein kinase Src is essential for macrophage-myofibroblast transition during renal scarring.

Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-ß/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-ß1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases.

Regulation of RhoA by STAT3 coordinates glial scar formation.

Understanding how the transcription factor signal transducer and activator of transcription-3 (STAT3) controls glial scar formation may have important clinical implications. We show that astrocytic STAT3 is associated with greater amounts of secreted MMP2, a crucial protease in scar formation. Moreover, we report that STAT3 inhibits the small GTPase RhoA and thereby controls actomyosin tonus, adhesion turnover, and migration of reactive astrocytes, as well as corralling of leukocytes in vitro. The inhibition of RhoA by STAT3 involves ezrin, the phosphorylation of which is reduced in STAT3-CKO astrocytes. Reduction of phosphatase and tensin homologue (PTEN) levels in STAT3-CKO rescues reactive astrocytes dynamics in vitro. By specific targeting of lesion-proximal, reactive astrocytes in Nestin-Cre mice, we show that reduction of PTEN rescues glial scar formation in Nestin-Stat3+/- mice. These findings reveal novel intracellular signaling mechanisms underlying the contribution of reactive astrocyte dynamics to glial scar formation.

Expression of mTOR/70S6K signaling pathway in pathological scar fibroblasts and the effects of resveratrol intervention.

The aim of the study was to examine the expression of mammalian target of rapamycin (mTOR)/70S6K signaling pathway in pathological scar fibroblasts and the effects of resveratrol (Res) intervention. The mTOR and 70S6K in pathological scar and normal skin fibroblasts were detected by immunofluorescence following treatment with different concentrations of Res. RT-PCR and western blot analysis were used to detect the expression of mTOR and 70S6K mRNA and protein, respectively. Immunofluorescence showed that the expression of 70S6K and mTOR was significantly enhanced in pathological scar fibroblasts, and mainly expressed in the nucleus, but not in normal skin fibroblasts. RT-PCR and western blot analysis showed that after different concentrations of Res treatments, the mTOR and 70S6K mRNA and protein expression significantly (P<0.05) decreased in a dose­dependent manner. In conclusion, the expression of mTOR/70S6K signaling pathway in pathological scar fibroblasts was significantly enhanced. Res can downregulate the expression of mTOR and 70S6K to achieve the inhibition of pathological scar fibroblast proliferation.

Aplicação da mitomicina C na cirurgia refrativa / Mitomycin C application in refractive surgery

RESUMO A mitomicina C teve seu uso profilático e terapêutico estabelecido, ao longo dos anos, para diminuir o haze depois da ablação superficial. A mitomicina C é segura e eficaz como uma terapia adjuvante aplicada após um procedimento primário de ceratectomia fotorrefrativa ou após um retratamento com ceratectomia fotorrefrativa após o laser in situ keratomileusis LASIK. A mitomicina age modulando a cicatrização após a cirurgia. Constitui-se num potente inibidor de mitose, bloqueia a ativação e a profliferação dos fibroblastos e a diferenciação dos miofibroblastos. Embora existam muitos estudos apontando a segurança da mitomicina nas doses ultilizadas, ainda persistem dúvidas quanto à segurança, a longo prazo, do uso da mitomicina. Quando as córneas são examinadas com microscópios confocal, após depleção inicial dos ceratócitos, a densidade celular parece retornar ao normal seis a 12 meses após o uso de mitomicina C . A maioria dos estudos clínicos não encontrou diferença significativa entre a densidade endotelial celular préoperatória e pós-operatória quando a mitomicina C 0.02% foi aplicada durante a cirurgia com um tempo de exposição de 2 minutos ou menos. Em aproximadamente 14 anos, a mitomicina C mostrou-se eficaz na prevenção e tratamento do haze corneano.

ABSTRACT Over the years, mitomycin C has been used by refractive surgeons to prophylactically decrease haze after surface ablation procedures and therapeutically in the treatment of preexisting haze. Development of mitomycin C treatments has had a significant role in the revival of surface ablation techniques. We reviewed the literature regarding mechanism of action of mitomycin C, its role in modulating wound healing after refractive surgery, and its safety and efficacy as adjuvant therapy applied after primary photorefractive keratectomy surgery or after photorefractive keratectomy re-treatment after laser in situ keratomileusis and other corneal surgeries and disorders. The drug is a potent mitotic inhibitor that effectively blocks keratocyte activation, proliferation, and myofibroblast differentiation. Many studies have suggested that mitomycin C is safe and effective in doses used by anterior surface surgeons, although there continue to be concerns regarding long-term safety. After initial depletion of anterior keratocytes, keratocyte density seems to return to normal 6 to12 months after the use of mitomycin C when corneas are examined with the confocal microscope. Most clinical studies found no difference between preoperative and postoperative corneal endothelial cell densities when mitomycin C 0.02% was applied during refractive surgery,with exposure time of 2 minutes or less. After approximately 14 years of use, mitomycin C has been found to be effective when used for prevention and treatment of corneal haze.

Evaluation of the Role of Granzyme B in Exuberant Scar Pathogenesis: An Immunohistochemical Study.

OBJECTIVE: To evaluate the possible role of Granzyme B (GzmB) in abnormal wound healing through its immunohistochemical expression in keloid and hypertrophic scars and to study the relationship of its expression with the clinicopathologic parameters of studied cases. STUDY DESIGN: Using immunohistochemical techniques, GzmB was analyzed in skin biopsies of 44 patients (30 cases with abnormal scars [21 with keloid and 9 with hypertrophic scars] and 14 age- and gender-matched cases with surgical scars as a control group). RESULTS: GzmB was expressed in keratinocytes in 28.6%, 66.7%, and 23.8% of surgical scars, hypertrophic scars, and keloids, respectively. Dermal expression in inflammatory cells was detected in 64.3%, 44.4%, and 28.6% of surgical scars, hypertrophic scars, and keloids, respectively. No significant difference was noted in GzmB expression intensity or percent in comparison between studied groups. CONCLUSION: GzmB plays no role in the pathogenesis of keloids and hypertrophic scars. Further large-scaled studies are needed to expand or refute the current observation.

Matrix remodeling by MMPs during wound repair.

Repair following injury involves a range of processes - such as re-epithelialization, scar formation, angiogenesis, inflammation, and more - that function, often together, to restore tissue architecture. MMPs carry out diverse roles in all of these activities. In this article, we discuss how specific MMPs act on ECM during two critical repair processes: re-epithelialization and resolution of scar tissue. For wound closure, we discuss how two MMPs - MMP1 in human epidermis and MMP7 in mucosal epithelia - facilitate re-epithelialization by cleaving different ECM or ECM-associated proteins to affect similar integrin:matrix adhesion. In scars and fibrotic tissues, we discuss that a variety of MMPs carry out a diverse range of activities that can either promote or limit ECM deposition. However, few of these MMP-driven activities have been demonstrated to be due a direct action on ECM.

Molecular mechanisms of scar-sourced axon growth inhibitors.

Astrogliosis is a defense response of the CNS to minimize primary damage and to repair injured tissues, but it ultimately generates harmful effects by upregulating inhibitory molecules to suppress neuronal elongation and forming potent barriers to axon regeneration. Chondroitin sulfate proteoglycans (CSPGs) are highly expressed by reactive scars and are potent contributors to the non-permissive environment in mature CNS. Surmounting strong inhibition by CSPG-rich scar is an important therapeutic goal for achieving functional recovery after CNS injuries. Currently, enzymatic digestion of CSPGs with locally applied chondroitinase ABC is the main in vivo approach to overcome scar inhibition, but several disadvantages may prevent using this bacterial enzyme as a therapeutic option for patients. A better understanding of molecular mechanisms underlying CSPG function may facilitate development of new effective therapies to overcome scar-mediated inhibition. Previous studies support that CSPGs act by non-specifically hindering the binding of matrix molecules to their cell surface receptors through steric interactions, but two members of the leukocyte common antigen related (LAR) phosphatase subfamily, protein tyrosine phosphatase σ and LAR, are functional receptors that bind CSPGs with high affinity and mediate CSPG inhibition. CSPGs may also act by binding two receptors for myelin-associated growth inhibitors, Nogo receptors 1 and 3. Thus, CSPGs inhibit axon growth through multiple mechanisms, making them especially potent and difficult therapeutic targets. Identification of CSPG receptors is not only important for understanding the scar-mediated growth suppression, but also for developing novel and selective therapies to promote axon sprouting and/or regeneration after CNS injuries. This article is part of a Special Issue entitled SI: Spinal cord injury.

[FUNCTIONING PROTEASES IN THE ESOPHAGUS MUCOSA AFTER CHEMICAL BURNS].

The main result of esophagus burn is the formation of scars, that caused by excessive synthesis of collagen and changes the balance of metalloproteinases and their tissue inhibitors. It was studied the activity of proteolytic enzymes, participation of MMP (metalloproteinase) and their tissue inhibitors (TIMP) in alkali burns of the esophagus 1st and 2nd degrees. We have shown a significant increase of TIMP level in homogenate after alkali burns of the esophagus (an average of 31-56% depend on of burn degree). We observed a reduced activity of serine proteinase after 1st degree burns on 15th, 21st day 35 and 18% respectively, after burns 2nd degree on 15th, 21st day 54 and 50%. The decrease of activity MMP after 1st degree burns on 15th and 21st day 30, 19%, respectively, in conditions of chemical burns 2nd degree on 15th and 21st day 30, 37%. These data may indicate the development of scarring after burn simulation of 2nd degree. Further investigation of the MMP and TIMP in the process of wound healing can be useful in creating effective approaches to prevent formation of post scarring of the esophagus.

Suberoylanilide hydroxamic acid (vorinostat): its role on equine corneal fibrosis and matrix metalloproteinase activity.

OBJECTIVE: To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. PROCEDURE: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFß1 to induce myofibroblast formation. The cultures were treated with either 5 µm or 10 µm SAHA for 72 h in the presence of TGFß1. Real-time PCR and immunocytochemistry were used to determine the antifibrotic efficacy of SAHA by quantifying α-smooth muscle actin (αSMA), a marker of myofibroblast formation and fibrosis. Real-time PCR was used to determine the effects of SAHA on MMP2 and MMP9 expression. Cytotoxicity of SAHA was evaluated with phase contrast microscopy and trypan blue exclusion assays. RESULTS: Suberoylanilide hydroxamic acid (SAHA) significantly attenuated TGFß1-induced differentiation of equine fibroblasts to myofibroblasts as indicated by 3- to 3.5-fold (P < 0.001) decrease in αSMA mRNA and 86-88% (P < 0.001) decrease in αSMA+ immunocytochemical staining. SAHA treatment also resulted in 4.5- to 5.5-fold (P < 0.01) decrease in MMP9 expression. A dose-dependent bimodal effect of SAHA on MMP2 expression was noted (3.5-fold increase with 5 µm dose; 0.5-fold decrease with 10 µm dose). No change in fibroblast viability was observed with a 5 µm SAHA dose, whereas a 10 µm dose resulted in a moderate 17% decrease in cell viability. CONCLUSIONS: Suberoylanilide hydroxamic acid (SAHA) can effectively inhibit TGFß-induced differentiation of equine corneal fibroblasts to myofibroblasts and modulates MMP production in vitro.

Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKα1.

Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodeling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodeling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase (TGF-ß1/p38 MAPK) pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodeling, as indicated by increased LV diastolic dimension 30days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.

Matrix metalloproteinase-28 deletion exacerbates cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2 macrophage activation.

RATIONALE: Matrix metalloproteinase (MMP)-28 regulates the inflammatory and extracellular matrix responses in cardiac aging, but the roles of MMP-28 after myocardial infarction (MI) have not been explored. OBJECTIVE: To determine the impact of MMP-28 deletion on post-MI remodeling of the left ventricle (LV). METHODS AND RESULTS: Adult C57BL/6J wild-type (n=76) and MMP null (MMP-28((-/-)), n=86) mice of both sexes were subjected to permanent coronary artery ligation to create MI. MMP-28 expression decreased post-MI, and its cell source shifted from myocytes to macrophages. MMP-28 deletion increased day 7 mortality because of increased cardiac rupture post-MI. MMP-28(-/-) mice exhibited larger LV volumes, worse LV dysfunction, a worse LV remodeling index, and increased lung edema. Plasma MMP-9 levels were unchanged in the MMP-28((-/-)) mice but increased in wild-type mice at day 7 post-MI. The mRNA levels of inflammatory and extracellular matrix proteins were attenuated in the infarct regions of MMP-28(-/-) mice, indicating reduced inflammatory and extracellular matrix responses. M2 macrophage activation was impaired when MMP-28 was absent. MMP-28 deletion also led to decreased collagen deposition and fewer myofibroblasts. Collagen cross-linking was impaired as a result of decreased expression and activation of lysyl oxidase in the infarcts of MMP-28(-/-) mice. The LV tensile strength at day 3 post-MI, however, was similar between the 2 genotypes. CONCLUSIONS: MMP-28 deletion aggravated MI-induced LV dysfunction and rupture as a result of defective inflammatory response and scar formation by suppressing M2 macrophage activation.

Calpain activity is essential in skin wound healing and contributes to scar formation.

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFß-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing.

Cutaneous wound healing is orchestrated by a number of physiological pathways that ultimately lead to reformation of skin integrity and the production of functional scar tissue. The remodeling of a wound is significantly affected by matrix metalloproteinases (MMPs), which act to control the degradation of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and, as such, have a role to play in the healing of infected wounds. For example, the zinc-metalloproteinase, elastase, produced by Pseudomonas aeruginosa, induces degradation of fibroblast proteins and proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. Microbial extracellular enzymes have also been shown to degrade human wound fluid and inhibit fibroblast cell growth. It is now being acknowledged that host and bacterial MMPs may act synergistically to cause tissue breakdown within the wound bed. Several studies have suggested that bacterial-derived secreted proteases may act to up-regulate the levels of MMPs produced by the host cells. Together, these findings indicate that bacterial phenotype in terms of protease producing potential of bacteria should be taken into consideration during diagnostic and clinical intervention of infected wound management. Furthermore, both host MMPs and those derived from infecting bacteria need to be targeted in order to increase the healing capacity of the injured tissue. The aim of this review is to investigate the evidence suggestive of a relationship between unregulated levels of both host and bacterial proteases and delayed wound healing.

Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing.

Connective Tissue Growth Factor (CTGF) and Transforming growth factor-ß1 (TGF-ß1) are key growth factors in regulating corneal scarring. Although CTGF was induced by TGF-ß1 and mediated many of fibroproliferative effects of TGF-ß1, the signaling pathway for CTGF production in corneal scarring remains to be clarified. In the present study, we firstly investigated the effects of c-Jun N-terminal kinase (JNK) on CTGF expression induce by TGF-ß1 in Telomerase-immortalized human cornea stroma fibroblasts (THSF). Then, we created penetrating corneal wound model and determined the effect of JNK in the pathogenesis of corneal scarring. TGF-ß1 activated MAPK pathways in THSF cells. JNK inhibitor significantly inhibited CTGF, fibronectin and collagen I expression induced by TGF-ß1 in THSF. In corneal wound healing, the JNK inhibitor significantly inhibited CTGF expression, markedly improved the architecture of corneal stroma and reduced corneal scar formation, but did not have a measurable impact on corneal wound healing in vivo. Our results indicate that JNK mediates the expression of CTGF and corneal scarring in corneal wound healing, and might be considered as specific targets of drug therapy for corneal scarring.

[Experimental study of the correlation of telomerase and intramedullary scar after spinal cord injury in rats].

OBJECTIVE: To detect the expression of telomerase in glial scar and its correlation with glial scar. METHODS: There were 120 Sprague Dawley rats were randomly divided into non-interference group of telomerase, interference group of telomerase and control group. Non-interference group and interference group were for spinal cord injury, which adopted Allen's Weight Dropping to make molding; control group was for sham operation to open the vertebral plate and expose spinal marrow, in which spinal cord injury would not be caused. The expression of telomerase and glial fibrillary acidic profein (GFAP) was detected by PCR-ELISA and Western blot, and the formation of glial scar was observed by immunofluorescence on the 1st, 3rd, 5th, 7th, 14th, 28th, 42 th and 56th day after the spinal injury, and analyzed its relativity. RESULTS: The expression of telomerase in non-interference group was (0.180 ± 0.004 - 1.217 ± 0.072), which was significantly higher than those in interference group (0.028 ± 0.007 - 0.092 ± 0.004, χ(2) = 28.753 - 37.518, P < 0.05) and control group (0.072 ± 0.007 - 0.075 ± 0.004, χ(2) = 18.618 - 41.093, P < 0.05) at all the time, with statistical significance. The expression of GFAP in non-interference group was (1.98 ± 0.15 - 19.40 ± 0.55) which was significantly higher than those in interference group (1.10 ± 0.13 - 16.64 ± 1.02, χ(2) = 14.538 - 37.366, P < 0.05) and control group (0.44 ± 0.05 - 0.48 ± 0.04, χ(2) = 16.733 - 34.041, P < 0.05) at all the time, with statistical significance. The expression of GFAP showed a linear correlation with that of telomerase in non-interference group, and with statistical differences (r = 0.755, P < 0.01). The expression of telomerase in interference group and control group were always negative. Glial scar observed by immunofluorescence in non-interference group was heavier than that in interference group, and control group showed no formation of glial scar. CONCLUSIONS: Telomerase shows a dynamic expression in glial scar and has positive correlational linear relationship with GFAP which shows the formation of glial scar. And the telomerase may be an important factor in promoting the formation of glial scar.

The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5.

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.