Dynamic Expression of Novel MiRNA Candidates and MiRNA-34 Family Members in Early- to Mid-Gestational Fetal Keratinocytes Contributes to Scarless Wound Healing by Targeting the TGF-β Pathway

BackgroundEarly- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. Keratinocytes (KCs) have been found to exert important effects on the regulation of fibroblasts. There may be significant differences of gestational fetal KCs at different ages. The advantages in early- to mid-gestational fetal KCs could lead to fetal scarless wound healing.MethodsKCs from six human fetal skin samples were divided into two groups: a mid-gestation group (less than 28 weeks of gestational age) and a late-gestation group (more than 28 weeks of gestational age). RNA extracted from KCs was used to prepare a library of small RNAs for next-generation sequencing (NGS). To uncover potential novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data. Other bioinformatical analyses were used to further validate the novel miRNAs. The expression levels of the miRNAs were further confirmed by real-time quantitative RT-PCR.ResultsA total of 61.59 million reads were mapped to 1,170 known human miRNAs in miRBase. Among a total of 202 potential novel miRNAs uncovered, 106 candidates have a higher probability of being novel human miRNAs. A total of 110 miRNAs, including 22 novel miRNA candidates, were significantly differently expressed between mid- and late-gestational fetal KCs. Thirty-three differentially expressed miRNAs and miR-34 family members are correlated with the transforming growth factor-β (TGF-β) pathway.ConclusionsTaken together, our results provide compelling evidence supporting the existence of 106 novel miRNAs and the dynamic expression of miRNAs that extensively targets the TGF-β pathway at different gestational ages in fetal KCs. MiRNAs showing altered expression at different gestational ages in fetal KCs may contribute to scarless wound healing in early- to mid-gestational fetal KCs, and thus may be new targets for potential scar prevention and reduction therapies.     

Plasminogen Activator Inhibitor-1 Regulates Integrin ��v��3 Expression and Autocrine Transforming Growth Factor �� Signaling

Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFβ inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease.Fibrotic disorders can result from environmental toxins, persistent infection, autoimmune disease, or mechanical injury, leading to the hardening and scarring of tissues. In fibrotic diseases, such as liver cirrhosis, renal fibrosis, and idiopathic lung fibrosis, or in pathological wound healing, such as hypertrophic scarring, scleroderma, and Dupuytren disease, the persistence of myofibroblasts contributes to disease progression by overproduction of extracellular matrix (ECM)² and by excessive contraction (1–3). A shift in the balance of growth factors and cytokines that promote ECM deposition and proteases that degrade matrix often contributes to fibrotic disease (4, 5). Plasmin, a broad spectrum protease that is generated from plasminogen by uPA, is one of the proteases that degrades matrix and activates growth factors and other proteases (6). Since uPA activity is inhibited by PAI-1, the overexpression of PAI-1 results in matrix accumulation. For this reason, PAI-1 is a key prognostic marker for fibrotic disease. PAI-1 exerts its inhibitory activity on uPA by stimulating the endocytosis of the cell surface uPA·uPAR complex through the low density lipoprotein receptor-related protein (7). Integrin αvβ3 is also internalized with the uPA·uPAR·low density lipoprotein receptor-related protein complex (8). After endocytosis, uPAR and integrins are recycled back to the cell surface for another round of binding (8, 9). uPAR and αvβ3 promote cellular attachment and spreading, since they are receptors for the extracellular matrix molecule, vitronectin (10). Thus, cycling of the complex is thought to stimulate the attachment and detachment that is necessary for cell migration (8). Consequently, a shift in the expression of any of these components (PAI-1/uPA/uPAR/αvβ3) can result in either aggressive migration, as seen in cancer invasion, or a persistent increase in cell adhesion and cell tension, as seen in myofibroblasts in fibrotic tissue.The family of TGFβ growth factors has been intensively studied for their role in fibrotic wound healing. Up-regulation of TGFβ results in amplified and persistent overproduction of molecules, such as integrins and PAI-1 and other protease inhibitors (e.g. TIMPs) (2, 3). Up-regulated integrins continue the cycle of TGFβ signaling by participating in the sustained activation of TGFβ from its latent form. To date, studies have found that various αv integrins participate in the activation of TGFβ (αvβ3, αvβ5, αvβ6, and αvβ8), but the mechanism differs (11–15). Integrins can serve as docking proteins to localize proteases that cleave and activate latent TGFβ in the ECM, or they can directly activate latent TGFβ in a protease-independent manner. Recently, it was discovered that latent TGFβ is also activated by mechanical stress generated from an integrin-mediated interaction between myofibroblasts and the ECM, primarily involving αvβ5. The mechanical stress promotes a conformational change that activates the latent TGFβ complex (15). αv integrins also modulate TGFβ signaling through the binding of αvβ3 to TGFβ receptor II (TGFβRII) in the presence of TGFβ. This interaction was shown to promote a dramatic increase in the proliferation of lung fibroblasts and induce invasion of epithelial breast cancer cells (16, 17).Our data establish a role for the PAI-1-mediated control of αvβ3 expression and support a significant role for αvβ3 in TGFβ signaling. Using PAI-1 KO cells, we tested the hypothesis that the absence of PAI-1 would result in the accumulation of αvβ3 on the cell surface, since PAI-1 promotes the endocytosis of uPA·uPAR·αvβ3. PAI-1-mediated endocytosis of β3 was significantly reduced in the PAI-1 KO cells. Correspondingly, we report that β3 accumulated at the cell surface in enlarged β3-containing focal adhesions. Thus, we explored whether the accumulation of αvβ3 on the cell surface had fibrogenic effects even in the absence of profibrotic PAI-1. Our results demonstrate dramatically increased TGFβ activity and an increase in collagen expression in PAI-1 KO cells. Together, these findings suggest that PAI-1 modulates β3 expression and localization and, in turn, TGFβ signaling. Our data reveal that maintaining precise levels of PAI-1 is a key to preventing fibrosis. Understanding the consequence of regulating PAI-1 activity is critical in light of the many clinical therapies currently under development that target PAI-1 (18, 19).     

Wound Healing Activity of Phage-Sisplayed TGF-β1 Model Peptide in Streptozotocin-Induced Diabetic Rats

International Journal of Peptide Research and Therapeutics - To evaluate the effect of phage-displayed TGF-β1 model peptide on cutaneous wound healing in streptozotocin-induced diabetic rats....     

Hydrocellular Foam Dressing Promotes Wound Healing along with Increases in Hyaluronan Synthase 3 and PPARα Gene Expression in Epidermis

Background

Hydrocellular foam dressing, modern wound dressing, induces moist wound environment and promotes wound healing: however, the regulatory mechanisms responsible for these effects are poorly understood. This study was aimed to reveal the effect of hydrocellular foam dressing on hyaluronan, which has been shown to have positive effects on wound healing, and examined its regulatory mechanisms in rat skin.

Methodology/Principal Findings

We created two full-thickness wounds on the dorsolateral skin of rats. Each wound was covered with either a hydrocellular foam dressing or a film dressing and hyaluronan levels in the periwound skin was measured. We also investigated the mechanism by which the hydrocellular foam dressing regulates hyaluronan production by measuring the gene expression of hyaluronan synthase 3 (Has3), peroxisome proliferator-activated receptor α (PPARα), and CD44. Hydrocellular foam dressing promoted wound healing and upregulated hyaluronan synthesis, along with an increase in the mRNA levels of Has3, which plays a primary role in hyaluronan synthesis in epidermis. In addition, hydrocellular foam dressing enhanced the mRNA levels of PPARα, which upregulates Has3 gene expression, and the major hyaluronan receptor CD44.

Conclusions/Significance

These findings suggests that hydrocellular foam dressing may be beneficial for wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis. We believe that the present study would contribute to the elucidation of the mechanisms underlying the effects of hydrocellular foam dressing-induced moist environment on wound healing and practice evidence-based wound care.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H₂O₂. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.     

Expression of α4 Integrin mRNA and Protein and Fibronectin in the Early Chicken Embryo

α₄ integrins (α₄β₁ and α₄β₇) have been shown to mediate both cell-matrix adhesion to fibronectin and cell-cell adhesion to VCAM-1. These interactions have been suggested to contribute to hematopoiesis, lymphocyte homing, recruitment of inflammatory cells, neural crest cell migration and myogenesis. We report here the cloning of chicken α₄ cDNA and its use to define the patterns of expression of α₄ mRNA and protein in early chicken embryos (19–22 somite pairs), a stage at which neural crest cells can be examined at various points in their migration and somitic development and differentiation can also be observed at various stages. We observe widespread expression of both α₄ mRNA and protein, although the patterns of steady state expression do not conform precisely. Many neural crest cells contain significant levels of α₄ mRNA. Some neural crest cells express α₄ protein but its expression is transient and/or limited to a subset of these cells. α₄ is strongly expressed at both mRNA and protein levels by somitic cells and their derivatives in the sclerotome, dermatome and myotome and is also expressed in neural tube, otic placode, heart, gut endoderm and some other tissues. Comparison with the distributions of fibronectin shows that, although some α₄ expression occurs in locations consistent with a role in cell-matrix adhesion to fibronectin, α₄ is also expressed in other places where fibronectin is low or absent and a role for α₄ in cell-cell interactions appears more likely.     

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

Rationale

Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and Results

In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion

Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.     

TGF-��1 and BRCA2 Expression are Associated with Clinical Factors in Breast Cancer

The objective of this study was to investigate the possible association between the expression of transforming growth factor beta-1 (TGF-β1) and breast cancer type2 susceptibility protein (BRCA2) with clinical factors in breast cancer. TGF-β1, BRCA2, human epidermal growth factor receptor2 (HER2), estrogen receptor, and progesterone receptor protein levels were measured in 67 samples from breast cancer patients by immunohistochemistry. The expression of these proteins was correlated with various clinical factors including age, pathohistological grade and status of axillary lymph node implication. TGF-β1 and BRCA2 were expressed in breast cancer tissues and expression of HER2 and TGF-β1 was significantly correlated with BRCA2. The authors conclude that elevated expression of BRCA2 correlates with TGF-β1 and HER2 in breast cancer and these three factors act in synergy to promote cancer. Thus, detection of both TGF-β1 and BRCA2 may therefore assist in the prognosis and treatment of breast cancer.     

PAR-3 Knockdown Enhances Adhesion Rate of PANC-1 Cells via Increased Expression of Integrinαv and E-Cadherin

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells'' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells'' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.     

Kefir Accelerates Burn Wound Healing Through Inducing Fibroblast Cell Migration In Vitro and Modulating the Expression of IL-1ß, TGF-ß1, and bFGF Genes In Vivo

Probiotics and Antimicrobial Proteins - Kefir is a natural probiotic compound with a long history of health benefits which can improve wound healing. This study investigated the regeneration...     

��v��3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.     

Correlations between Integrin ανβ6 Expression and Clinico-Pathological Features in Stage B and Stage C Rectal Cancer

Integrin ανβ6 is highly expressed in a range of human cancers and frequently correlates with patient survival. This study examines correlations between ανβ6 expression and patient clinico-pathological features in Stage B and Stage C rectal cancer, including overall survival. Expression of ανβ6 was measured in 362 Stage B or C rectal cancer tissue samples at the tumour central region, invasive tumour front and adjacent non-neoplastic mucosa using immunohistochemistry. Distribution of ανβ6 was found to be significantly higher at the invasive front compared to central regions of the tumour (p<0.001) or adjacent non-neoplastic mucosa (p<0.001) suggesting ανβ6 plays a role in tumour cell invasion. However, integrin ανβ6 expression was not associated with clinico-pathological features or overall survival indicating it is not an independent prognostic marker differentiating Stage B or C rectal cancer. Previous ανβ6 studies have suggested the expression of ανβ6 is involved in the earlier stages (i.e. Stages A/B) of tumour progression rather than the later stages (i.e. Stages C/D). However, our study has revealed that in rectal cancer ανβ6 expression does not increase between Stages B and C, but may occur earlier, namely before or during Stage B cancer.     

Corneal Wound Healing Requires IKB kinase β Signaling in Keratocytes

IkB kinase β (IKKβ) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKKβ in the cornea using Ikkβ^ΔCS mice in which the Ikkβ gene was specifically deleted in the corneal stromal keratocytes. The Ikkβ^ΔCS corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the Ikkβ^F/F corneas that restored transparency in 2 weeks after injury, over 50% of the Ikkβ^ΔCS corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased α smooth muscle actin (α-SMA) expression, higher levels of senescence β-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the Ikkβ^ΔCS corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKKβ in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.     

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.