Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process.In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3.Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.     

Maternal hypoxia alters matrix metalloproteinase expression patterns and causes cardiac remodeling in fetal and neonatal rats

Fetal hypoxia leads to progressive cardiac remodeling in rat offspring. The present study tested the hypothesis that maternal hypoxia results in reprogramming of matrix metalloproteinase (MMP) expression patterns and fibrillar collagen matrix in the developing heart. Pregnant rats were treated with normoxia or hypoxia (10.5% O(2)) from day 15 to 21 of gestation. Hearts were isolated from 21-day fetuses (E21) and postnatal day 7 pups (PD7). Maternal hypoxia caused a decrease in the body weight of both E21 and PD7. The heart-to-body weight ratio was increased in E21 but not in PD7. Left ventricular myocardium wall thickness and cardiomyocyte proliferation were significantly decreased in both fetal and neonatal hearts. Hypoxia had no effect on fibrillar collagen content in the fetal heart, but significantly increased the collagen content in the neonatal heart. Western blotting revealed that maternal hypoxia significantly increased collagen I, but not collagen III, levels in the neonatal heart. Maternal hypoxia decreased MMP-1 but increased MMP-13 and membrane type (MT)1-MMP in the fetal heart. In the neonatal heart, MMP-1 and MMP-13 were significantly increased. Active MMP-2 and MMP-9 levels and activities were not altered in either fetal or neonatal hearts. Hypoxia significantly increased tissue inhibitors of metalloproteinase (TIMP)-3 and TIMP-4 in both fetal and neonatal hearts. In contrast, TIMP-1 and TIMP-2 were not affected. The results demonstrate that in utero hypoxia reprograms the expression patterns of MMPs and TIMPs and causes cardiac tissue remodeling with the increased collagen deposition in the developing heart.     

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.     

Scarless skin wound healing in FOXN1 deficient (nude) mice is associated with distinctive matrix metalloproteinase expression

Similar to mammalian fetuses FOXN1 deficient (nude) mice are able to restore the structure and integrity of injured skin in a scarless healing process by mechanisms independent of the genetic background. Matrix metalloproteinases (MMPs) are required for regular skin wound healing and the distinctive pattern of their expression has been implicated to promote scarless healing. In this study, we analyzed the temporal and spatial expression patterns of these molecules during the incisional skin wounds in adult nude mice. Macroscopic and histological analyses of skin wounds revealed an accelerated wound healing process, minimal granulation tissue formation and markedly diminished scarring in nude mice. Quantitative RT-PCR (Mmp-2, -3, -8, -9, -10, -12, -13, -14 and Timp-1, -2, -3), Western blots (MMP-13) and gelatin zymography (MMP-9) revealed that MMP-9 and MMP-13 showed a unique, bimodal pattern of up-regulation during the early and late phases of wound healing in nude mice. Immunohistochemically MMP-9 and MMP-13 were generally detected in epidermis during the early phase and in dermis during the late (remodeling) phase. Consistent with these in vivo observations, dermal fibroblasts cultured from nude mice expressed higher levels of types I and III collagen, MMP-9 and MMP-13 mRNA levels and higher MMP enzyme activity than wild type controls. Collectively, these finding suggest that the bimodal pattern of MMP-9 and MMP-13 expression during skin repair process in nude mice could be a major component of their ability for scarless healing.     

Scarless fetal wound healing: a basic science review

SUMMARY: Scar formation is a major medical problem that can have devastating consequences for patients. The adverse physiological and psychological effects of scars are broad, and there are currently no reliable treatments to prevent scarring. In contrast to adult wounds, early gestation fetal skin wounds repair rapidly and in the absence of scar formation. Despite extensive investigation, the exact mechanisms of scarless fetal wound healing remain largely unknown. For some time, it has been known that significant differences exist among the extracellular matrix, inflammatory response, cellular mediators, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in scarless wound repair.     

Imbalances between Matrix Metalloproteinases (MMPs) and Tissue Inhibitor of Metalloproteinases (TIMPs) in Maternal Serum during Preterm Labor

Background

Matrix metalloproteinases (MMPs) are involved in remodeling of the extracellular matrix (ECM) during pregnancy and parturition. Aberrant ECM degradation by MMPs or an imbalance between MMPs and their tissue inhibitors (TIMPs) have been implicated in the pathogenesis of preterm labor, however few studies have investigated MMPs or TIMPs in maternal serum. Therefore, the purpose of this study was to determine serum concentrations of MMP-3, MMP-9 and all four TIMPs as well as MMP:TIMP ratios during term and preterm labor.

Methods

A case control study with 166 singleton pregnancies, divided into four groups: (1) women with preterm birth, delivering before 34 weeks (PTB); (2) gestational age (GA) matched controls, not in preterm labor; (3) women at term in labor and (4) at term not in labor. MMP and TIMP concentrations were measured using Luminex technology.

Results

MMP-9 and TIMP-4 concentrations were higher in women with PTB vs. GA matched controls (resp. p = 0.01 and p<0.001). An increase in MMP-9:TIMP-1 and MMP-9:TIMP-2 ratio was observed in women with PTB compared to GA matched controls (resp. p = 0.02 and p<0.001) as well as compared to women at term in labor (resp. p = 0.006 and p<0.001). Multiple regression results with groups recoded as three key covariates showed significantly higher MMP-9 concentrations, higher MMP-9:TIMP-1 and MMP-9:TIMP-2 ratios and lower TIMP-1 and -2 concentrations for preterm labor. Significantly higher MMP-9 and TIMP-4 concentrations and MMP-9:TIMP-2 ratios were observed for labor.

Conclusions

Serum MMP-9:TIMP-1 and MMP-9:TIMP-2 balances are tilting in favor of gelatinolysis during preterm labor. TIMP-1 and -2 concentrations were lower in preterm gestation, irrespective of labor, while TIMP-4 concentrations were raised in labor. These observations suggest that aberrant serum expression of MMP:TIMP ratios and TIMPs reflect pregnancy and labor status, providing a far less invasive method to determine enzymes essential in ECM remodeling during pregnancy and parturition.     

Expression profiles of collagens,HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic, uncommon, sub-epidermal blistering disease involving the skin and mucous membranes that heals with scar formation and milia. Collagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important components that play an essential role(s) in matrix remodeling during scar formation. However, the possible involvement of these components in EBA-induced scarring is not yet known. In the present study, we examined the expression profile of collagens, collagen-binding heat shock protein 47 (HSP47), MMPs and their inhibitory enzymes, TIMPs, in matrix remodeling during conjunctival scarring. The involvement of TGF-beta1, a fibrogenic factor, was also studied. Compared to the controls, an increased expression of type I collagen, type III collagen and HSP47 was detected in conjunctival biopsy sections of patient with EBA using immunohistochemistry. Similar increase in the expression of type I collagen, type III collagen and HSP47 was noted in conjunctival fibroblasts obtained from the patient with EBA. Up-regulation in the expression of MMP-1 and MMP-14 was also noted in conjunctival fibroblasts isolated from the patient with EBA, while no significant changes in the expression of MMP-3, MMP-8, MMP-9 and MMP-13 were seen. As for TIMPs, conjunctival fibroblasts isolated from the patient with EBA, grown in vitro, exhibited increased expression of TIMP-1, TIMP-2 and TIMP-3, when compared with fibroblasts grown from control conjunctival tissues, although the expression level varies with different molecules of the same family. Additionally, compared to the control conjunctival fibroblasts, an increased expression of TGF-beta1 was detected in fibroblasts isolated from the conjunctival tissues of patient with EBA.This study suggests that there is up-regulation in the production of collagens (type I and III), collagen-binding protein (HSP47), matrix degrading collagenases (MMP-1 and 14), and their inhibitory enzymes (TIMP-1, 2 and 3) during the process of conjunctival matrix remodeling in the patient with EBA. The presented data is preliminary and could serve as a basis for further studies to enhance our understanding about the molecular mechanisms of conjunctival scarring in patients with EBA.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Scarless integumentary wound healing in the mammalian fetus: Molecular basis and therapeutic implications

Adult mammals respond to injury of their skin/integument by forming scar tissue. Scar is useful in rapidly sealing an injured area, but can also lead to significant morbidity. Mammals in fetal life retain the ability to heal integumentary wounds regeneratively, without scar. The critical molecular mechanisms governing this remarkable phenomenon have been a subject of great interest, in the hopes that these could be dissected and recapitulated in the healing adult wound, with the goal of inducing scarless healing in injured patients. Multiple lines of investigation spanning decades have implicated a number of factors in distinguishing scarless from fibrotic wound healing, including most prominently transforming growth factor‐β and interleukin‐10, among others. Therapeutic interventions to try to mitigate scarring in adult wounds have been developed out of these studies, and have reached the level of clinical trials in humans, although as yet no FDA‐approved treatment exists. More recent expressomic studies have revealed many more genes that are differentially expressed in scarlessly healing fetal wounds compared with adult, and microRNAs have also been identified as participating in the fetal wound healing response. These represent an even greater range of potential therapeutics (or targets for therapy) to translate the promise of scarless fetal wound healing to the injured adult patient. Birth Defects Research (Part C) 96:223–236, 2012. © 2012 Wiley Periodicals, Inc.     

MMP-2、MMP-7、MMP-9、TIMP-1及TIMP-2在乳腺癌组织中的表达及意义

目的:探讨基质金属蛋白酶及其抑制剂在乳腺癌组织中的表达及其与肿瘤浸润转移的关系,为乳腺癌的临床治疗及预后预测提供基础。方法:选择我院2012年5月至2014年5月收治的乳腺癌患者80例,对所选病例的乳腺癌组织、癌旁组织及正常乳腺组织样本进行检测。观察并比较不同乳腺组织中MMP-2,MMP-7、MMP-9、TIMP-1及TIMP-2 m RNA的表达水平。结果:与正常乳腺组织相比较,乳腺癌组织和癌旁组织中MMP-2、MMP-7、MMP-9,TIMP-1及TIMP-2 m RNA的表达显著增加,差异具有统计学意义(P0.05)。乳腺癌组织中MMP-2、MMP-7、MMP-9、TIMP-1及TIMP-2 m RNA的表达显著高于癌旁组织和正常组织,差异具有统计学意义(P0.05)。随着肿瘤范围扩大,MMP-2、MMP-7和MMP-9 m RNA的表达水平显著增加(P0.05),而TIMP-1和TIMP-2 m RNA表达无显著变化(P0.05)。随着淋巴结转移进展,MMP-2、MMP-7和MMP-9 m RNA的表达显著增加(P0.05),而TIMP-1和TIMP-2 m RNA无显著变化(P0.05)。结论:MMP-2、MMP-7、MMP-9、TIMP-1和TIMP-2的m RNA在乳腺癌组织中呈高表达,这可能与乳腺癌的发生和发展有关,而MMP-2、MMP-7和MMP-9可能有助于预测乳腺癌的侵袭行为。     

Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts

Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The goal of this study was to elucidate peculiarities of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF). The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papillomavirus (HPV-16). Papillomaviruses type16 and 18 are the etiological factor for cervical cancer. A primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF included determination of MMP-2 and MMP-9 activity by hydrolysis of the specific substrate, radioactive collagen type IV; analysis of MMP spectra by a zymographic assay, and estimation of the mRNA expression by RT-PCR. It was found that: (1) collagenolytic activity of MMP was increased only in TF and it depended on the degree of cell tumorigenicity; (2) the study of MMP spectra revealed the presence of MMP-9 only in TF, whereas MMP-2 was found in IF as well; (3) the mRNA expression of MMP-9, MT1-MMP and TIMP-2 increased in all TF while the MMP-2 expression increased in TF only after TF cell selection on rats; (4) the collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 and endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to the PF culture. The data obtained indicate changes in the ratio enzyme/activator/inhibitor and also suggest a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV16 gene in a cell culture.     

Hydrogen sulfide protects against vascular remodeling from endothelial damage

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H₂S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT + NaHS (sodium hydrogen sulfide, precursor of H₂S) treatment (30 μmol/L in drinking water/6 weeks) and WTD + NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H₂S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H₂S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.