Tripterygium wilfordii derivative celastrol, a YAP inhibitor, has antifibrotic effects in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by extensive tissue fibrosis maintained by mechanotranductive/proadhesive signalling. Drugs targeting this pathway are therefore of likely therapeutic benefit. The mechanosensitive transcriptional co-activator, yes activated protein-1 (YAP1), is activated in SSc fibroblasts. The terpenoid celastrol is a YAP1 inhibitor; however, if celastrol can alleviate SSc fibrosis is unknown. Moreover, the cell niches required for skin fibrosis are unknown. METHODS: Human dermal fibroblasts from healthy individuals and patients with diffuse cutaneous SSc were treated with or without transforming growth factor ß1 (TGFß1), with or without celastrol. Mice were subjected to the bleomycin-induced model of skin SSc, in the presence or absence of celastrol. Fibrosis was assessed using RNA Sequencing, real-time PCR, spatial transcriptomic analyses, Western blot, ELISA and histological analyses. RESULTS: In dermal fibroblasts, celastrol impaired the ability of TGFß1 to induce an SSc-like pattern of gene expression, including that of cellular communication network factor 2, collagen I and TGFß1. Celastrol alleviated the persistent fibrotic phenotype of dermal fibroblasts cultured from lesions of SSc patients. In the bleomycin-induced model of skin SSc, increased expression of genes associated with reticular fibroblast and hippo/YAP clusters was observed; conversely, celastrol inhibited these bleomycin-induced changes and blocked nuclear localisation of YAP. CONCLUSIONS: Our data clarify niches within the skin activated in fibrosis and suggest that compounds, such as celastrol, that antagonise the YAP pathway may be potential treatments for SSc skin fibrosis.

Human severe sepsis cytokine mixture increases ß2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro.

INTRODUCTION: Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. METHODS: The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)). RESULTS: Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either ß2 (CD18), αL/ß2 (CD11α/CD18; LFA-1) or αM/ß2 (CD11ß/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. CONCLUSIONS: Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a ß2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

LipidSeq: a next-generation clinical resequencing panel for monogenic dyslipidemias.

We report the design of a targeted resequencing panel for monogenic dyslipidemias, LipidSeq, for the purpose of replacing Sanger sequencing in the clinical detection of dyslipidemia-causing variants. We also evaluate the performance of the LipidSeq approach versus Sanger sequencing in 84 patients with a range of phenotypes including extreme blood lipid concentrations as well as additional dyslipidemias and related metabolic disorders. The panel performs well, with high concordance (95.2%) in samples with known mutations based on Sanger sequencing and a high detection rate (57.9%) of mutations likely to be causative for disease in samples not previously sequenced. Clinical implementation of LipidSeq has the potential to aid in the molecular diagnosis of patients with monogenic dyslipidemias with a high degree of speed and accuracy and at lower cost than either Sanger sequencing or whole exome sequencing. Furthermore, LipidSeq will help to provide a more focused picture of monogenic and polygenic contributors that underlie dyslipidemia while excluding the discovery of incidental pathogenic clinically actionable variants in nonmetabolism-related genes, such as oncogenes, that would otherwise be identified by a whole exome approach, thus minimizing potential ethical issues.

Inhibition of focal adhesion kinase prevents experimental lung fibrosis and myofibroblast formation.

OBJECTIVE: Enhanced adhesive signaling, including activation of focal adhesion kinase (FAK), is a hallmark of fibroblasts from lung fibrosis patients, and FAK has therefore been hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro. METHODS: FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor PF-562,271, or with small interfering RNA (siRNA)-mediated silencing of FAK were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and lungs were harvested for histologic and biochemical analysis. Using endothelin 1 (ET-1) as a stimulus, cell adhesion and contraction, as well as profibrotic gene expression, were studied in fibroblasts isolated from wild-type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild-type and ß1 integrin-deficient mouse fibroblasts. RESULTS: FAK expression and activity were up-regulated in fibroblast foci and remodeled vessels from lung fibrosis patients. Pharmacologic or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis in mice. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by JNK activation through ß1 integrin/FAK signaling. CONCLUSION: These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

FAK is required for TGFbeta-induced JNK phosphorylation in fibroblasts: implications for acquisition of a matrix-remodeling phenotype.

Transforming growth factor beta (TGFbeta) plays a critical role in connective tissue remodeling by fibroblasts during development, tissue repair, and fibrosis. We investigated the molecular pathways in the transmission of TGFbeta signals that lead to features of connective tissue remodeling, namely formation of an alpha-smooth muscle actin (alpha-SMA) cytoskeleton, matrix contraction, and expression of profibrotic genes. TGFbeta causes the activation of focal adhesion kinase (FAK), leading to JNK phosphorylation. TGFbeta induces JNK-dependent actin stress fiber formation, matrix contraction, and expression of profibrotic genes in fak+/+, but not fak-/-, fibroblasts. Overexpression of MEKK1, a kinase acting upstream of JNK, rescues TGFbeta responsiveness of JNK-dependent transcripts and actin stress fiber formation in FAK-deficient fibroblasts. Thus we propose a FAK-MEKK1-JNK pathway in the transmission of TGFbeta signals leading to the control of alpha-SMA cytoskeleton reorganization, matrix contraction, and profibrotic gene expression and hence to the physiological and pathological effects of TGFbeta on connective tissue remodeling by fibroblasts.

Transcriptional profiling of leukocytes in critically ill COVID19 patients: implications for interferon response and coagulation.

BACKGROUND: COVID19 is caused by the SARS-CoV-2 virus and has been associated with severe inflammation leading to organ dysfunction and mortality. Our aim was to profile the transcriptome in leukocytes from critically ill patients positive for COVID19 compared to those negative for COVID19 to better understand the COVID19-associated host response. For these studies, all patients admitted to our tertiary care intensive care unit (ICU) suspected of being infected with SARS-CoV-2, using standardized hospital screening methodologies, had blood samples collected at the time of admission to the ICU. Transcriptome profiling of leukocytes via ribonucleic acid sequencing (RNAseq) was then performed and differentially expressed genes as well as significantly enriched gene sets were identified. RESULTS: We enrolled seven COVID19 + (PCR positive, 2 SARS-CoV-2 genes) and seven age- and sex-matched COVID19- (PCR negative) control ICU patients. Cohorts were well-balanced with the exception that COVID19- patients had significantly higher total white blood cell counts and circulating neutrophils and COVID19 + patients were more likely to suffer bilateral pneumonia. The mortality rate for this cohort of COVID19 + ICU patients was 29%. As indicated by both single-gene based and gene set (GSEA) approaches, the major disease-specific transcriptional responses of leukocytes in critically ill COVID19 + ICU patients were: (i) a robust overrepresentation of interferon-related gene expression; (ii) a marked decrease in the transcriptional level of genes contributing to general protein synthesis and bioenergy metabolism; and (iii) the dysregulated expression of genes associated with coagulation, platelet function, complement activation, and tumour necrosis factor/interleukin 6 signalling. CONCLUSIONS: Our findings demonstrate that critically ill COVID19 + patients on day 1 of admission to the ICU display a unique leukocyte transcriptional profile that distinguishes them from COVID19- patients, providing guidance for future targeted studies exploring novel prognostic and therapeutic aspects of COVID19.

Hyperactive TORC1 sensitizes yeast cells to endoplasmic reticulum stress by compromising cell wall integrity.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). Here, we investigated how the target of rapamycin complex 1 (TORC1) signaling cascade acts in parallel with the UPR to regulate ER stress sensitivity. Using Saccharomyces cerevisiae, we found that TORC1 signaling is attenuated during ER stress and constitutive activation of TORC1 increases sensitivity to ER stressors independently of the UPR. Transcriptome analysis revealed that TORC1 hyperactivation results in cell wall remodelling. Conversely, hyperactive TORC1 sensitizes cells to cell wall stressors, including the antifungal caspofungin. Elucidating the crosstalk between the UPR, cell wall integrity, and TORC1 signaling may uncover new paradigms through which the response to protein misfolding is regulated.

Fibroblast adhesion results in the induction of a matrix remodeling gene expression program.

Fibrosis is believed to occur through the failure to terminate the normal tissue remodeling program. Tissue repair intimately involves the ability of fibroblasts to attach to extracellular matrix (ECM), resulting in cell migration and ECM contraction. Elevated, activated adhesive signaling is a key phenotypic hallmark of fibrotic cells. The precise contribution of adhesion to tissue remodeling and repair and fibrotic responses in fibroblasts is unclear, but involves focal adhesion kinase (FAK). FAK signals downstream of integrin-mediates attachment of fibroblasts to extracellular matrix. In this report, we show that FAK is required for the expression of a cohort of mRNAs encoding ECM and matrix remodeling genes including CCN2, alpha-smooth muscle actin (SMA) and type I collagen. Adhesion of fibroblasts to fibronectin, a component of the provisional matrix deposited in the initial phases of tissue repair, also resulted in the induction of CCN2, alpha-SMA and type I collagen mRNAs. Endothelin-1 (ET-1), a key inducer of pro-fibrotic gene expression, was also induced upon fibroblast attachment to ECM, and antagonism of the ET-1 receptors significantly reduced the ability of adhesion to induce expression of CCN2, alpha-SMA and type I collagen mRNAs. These results suggest that adhesion of fibroblasts to matrix during the initial phases of tissue remodeling and repair may actively contribute to the tissue repair program through the induction of pro-fibrotic gene expression.

Glucose dependence of the regulated secretory pathway in alphaTC1-6 cells.

We have investigated the effects of chronically elevated glucose concentrations on the pancreatic alpha-cell line alphaTC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2. The secretion of GLP-1, a proglucagon-derived peptide produced by cleavage of proglucagon by PC1/3, was also increased in response to high glucose. Gene expression profiling experiments showed that a number of components of the regulated secretory pathway were up-regulated at high glucose concentrations, including processing enzymes and exocytotic proteins. Immunoblot analysis showed that the expression of the exocytotic SNARE proteins, as well as that of PC1/3, chromogranin A, and 7B2, were all increased after chronic exposure to high glucose levels. Immunocytochemistry showed no changes in the expression of the mature alpha-cell markers glucagon and brn-4 and no induction of the immature alpha-cell marker pdx-1. We conclude that chronically elevated glucose concentrations up-regulate the regulated secretory response of the alpha-cell.

Quality assessment of microarray experiments.

OBJECTIVES: We describe a quality assurance procedure to maximize the value of oligonucleotide microarray expression profiles. DESIGN, METHODS, AND RESULTS: Background microarray noise was 82.2+/-54.5 and 51.8+/-12.4 units, respectively, before and after enacting the program (P<0.0001). We also noted improved concordance of microarray expression fold-changes for selected genes with results of RT-PCR validation. CONCLUSIONS: This multi-step procedure, including quantification of RNA sample degradation and detection of outlier data points, has increased data quality from our microarray facility.

CCN2 Expression by Tumor Stroma Is Required for Melanoma Metastasis.

Metastatic melanoma has an extremely poor prognosis with few durable remissions. The secreted matricellular protein connective tissue growth factor (CCN2) is overexpressed in cancers including melanoma and may represent a viable therapeutic target. However, the mechanism underlying the contribution of CCN2 to melanoma progression is unclear. Herein, we use the highly metastatic murine melanoma cell line B16(F10) and syngeneic mice, in which CCN2 expression is knocked out in fibroblasts, to demonstrate that loss of CCN2, either in melanoma cells or in the niche, impedes the ability of melanoma cells to invade. Specifically, loss of CCN2 in melanoma cells diminished their ability to invade through collagen in vitro, and loss of fibroblast-derived CCN2 decreased spontaneous metastases of melanoma cells from the skin to the lungs in vivo. Proliferation and tumor growth were not affected by loss of CCN2. CCN2-deficient B16(F10) cells showed reduced expression of the matricellular protein periostin; addition of recombinant periostin rescued the in vitro invasion defect of these cells. Immunohistochemical analysis of CCN2-deficient mice confirmed loss of periostin expression in the absence of CCN2. CCN2 and periostin mRNA levels are positively correlated with each other and with the stromal composition of human melanoma lesions but not BRAF mutations. Thus, CCN2 promotes invasion and metastasis via periostin and should be further evaluated as a possible therapeutic target for BRAF inhibitor-resistant melanoma.

Optimizing RNA extraction yield from whole blood for microarray gene expression analysis.

OBJECTIVES: Microarray analysis of gene expression profiles of blood leukocytes has many potential clinical and research applications. DESIGN AND METHODS: We used the PAXgene Blood RNA System to prepare RNA from the whole blood of normal volunteers using two incubation times followed by gene expression profiling using the Affymetrix HU133A GeneChip. CONCLUSIONS: Longer incubation gave a significantly higher RNA yield and samples that were satisfactory for microarray analysis, with excellent pairwise correlations between replicates.

miR-218 regulates focal adhesion kinase-dependent TGFß signaling in fibroblasts.

Scarring, which occurs in essentially all adult tissue, is characterized by the excessive production and remodeling of extracellular matrix by α-smooth muscle actin (SMA)-expressing myofibroblasts located within connective tissue. Excessive scarring can cause organ failure and death. Oral gingivae do not scar. Compared to dermal fibroblasts, gingival fibroblasts are less responsive to transforming growth factor ß (TGFß) due to the reduced expression, due to the reduced expression and activity of focal adhesion kinase (FAK) by this cell type. Here we show that, compared with dermal fibroblasts, gingival fibroblasts show reduced expression of miR-218. Introduction of pre-miR-218 into gingival fibroblasts elevates FAK expression and, via a FAK/src-dependent mechanism, results in the ability of TGFß to induce α-SMA. The deubiquitinase cezanne is a direct target of miR-218 and has increased expression in gingival fibroblasts compared with dermal fibroblasts. Knockdown of cezanne in gingival fibroblasts increases FAK expression and causes TGFß to induce α-smooth muscle actin (α-SMA). These results suggest that miR-218 regulates the ability of TGFß to induce myofibroblast differentiation in fibroblasts via cezanne/FAK.

TAK1 is required for dermal wound healing and homeostasis.

Dermal connective tissue is a supportive structure required for skin's barrier function; dysregulated dermal homeostasis results in chronic wounds and fibrotic diseases. The multifunctional cytokine transforming growth factor (TGF) ß promotes connective tissue deposition, repair, and fibrosis. TGF-ß acts through well-defined canonical pathways; however, the non-canonical pathways through which TGF-ß selectively promotes connective tissue deposition are unclear. In dermal fibroblasts, we show that inhibition of the non-canonical TGF-ß-activated kinase 1 (TAK1) selectively reduced the ability of TGF-ß to induce expression of a cohort of wound healing genes, such as collagens, CCN2, TGF-ß1, and IL-6. Fibroblast-specific TAK1-knockout mice showed impaired cutaneous tissue repair and decreased collagen deposition, α-smooth muscle actin and CCN2 expression, proliferating cell nuclear antigen staining, and c-Jun N-terminal kinase and p38, but not Smad3, phosphorylation. TAK1-deficient fibroblasts showed reduced cell proliferation, migration, cell attachment/spreading, and contraction of a floating collagen gel matrix. TAK1-deficient mice also showed progressively reduced skin thickness and collagen deposition. Thus, TAK1 is essential for connective tissue deposition in the dermis.

Cellular mechanisms underlying failed beta cell regeneration in offspring of protein-restricted pregnant mice.

Low birth weight and poor foetal growth following low protein (LP) exposure are associated with altered islet development and glucose intolerance in adulthood. Additionally, LP-fed offspring fail to regenerate their ß-cells following depletion with streptozotocin (STZ) in contrast to control-fed offspring that restore ß-cell mass. Our objective was to identify signalling pathways and cellular functions that may be critically altered in LP offspring rendering them susceptible to developing long-term glucose intolerance and decreased ß-cell plasticity. Pregnant Balb/c mice were fed a control (C; 20% protein) or an isocaloric LP (8% protein) diet throughout gestation and C diet thereafter. Female offspring were injected intraperitoneally with 35 mg/kg STZ or vehicle on days 1 to 5 for each dietary treatment. At 30 days of age, total RNA was extracted from pancreatic tissue for microarray analysis using the Affymetrix GeneChip Mouse Genome 430 2.0. Gene and protein expression were quantified from isolated islets. Finally, ß-cell proliferation was determined in vitro following REG1α treatment. The microarray data and GO enrichment analysis indicated that foetal protein restriction alters the early expression of genes necessary for many cell functions, such as oxidative phosphorylation and free radical scavenging. Expression of Reg1 was upregulated following STZ, whereas protein content was decreased in LP + STZ islets. Furthermore, REG1α failed to stimulate ß-cell proliferation in vitro in LP + STZ islets. Therefore, early nutritional insults may programme the Reg1 pathway resulting in a limited ability to increase ß-cell mass during metabolic stress. In conclusion, this study implicates the Reg1 pathway in ß-cell regeneration and describes altered programming of gene expression in LP offspring, which underlies later development of cell dysfunction and glucose intolerance in adulthood.

RanBPM expression regulates transcriptional pathways involved in development and tumorigenesis.

RanBPM is a ubiquitous protein that has been reported to regulate several cellular processes through interactions with various proteins. However, it is not known whether RanBPM may regulate gene expression patterns. As it has been shown that RanBPM interacts with a number of transcription factors, we hypothesized that it may have wide ranging effects on gene expression that may explain its function. To test this hypothesis, we generated stable RanBPM shRNA cell lines to analyze the effect of RanBPM on global gene expression. Microarray analyses were conducted comparing the gene expression profile of Hela and HCT116 RanBPM shRNA cells versus control shRNA cells. We identified 167 annotated genes significantly up- or down-regulated in the two cell lines. Analysis of the gene set revealed that down-regulation of RanBPM led to gene expression changes that affect regulation of cell, tissue, and organ development and morphology, as well as biological processes implicated in tumorigenesis. Analysis of Transcription Factor Binding Sites (TFBS) present in the gene set identified several significantly over-represented transcription factors of the Forkhead, HMG, and Homeodomain families of transcription factors, which have previously been demonstrated as having important roles in development and tumorigenesis. In addition, the combined results of these analyses suggested that several signaling pathways were affected by RanBPM down-regulation, including ERK1/2, Wnt, Notch, and PI3K/Akt pathways. Lastly, analysis of selected target genes by quantitative RT-qPCR confirmed the changes revealed by microarray. Several of the genes up-regulated in RanBPM shRNA cells encode proteins with known oncogenic functions, such as the RON tyrosine kinase, the adhesion molecule L1CAM, and transcription factor ELF3/ESE-1, suggesting that RanBPM functions as a tumor suppressor to prevent deregulated expression of these genes. Altogether, these results suggest that RanBPM does indeed function to regulate many genomic events that regulate embryonic, tissue, and cellular development as well as those involved in cancer development and progression.

Multiple roles of integrin-linked kinase in epidermal development, maturation and pigmentation revealed by molecular profiling.

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.

An analysis of the myocardial transcriptome in a mouse model of cardiac dysfunction with decreased cholinergic neurotransmission.

Autonomic dysfunction is observed in many cardiovascular diseases and contributes to cardiac remodeling and heart disease. We previously reported that a decrease in the expression levels of the vesicular acetylcholine transporter (VAChT) in genetically-modified homozygous mice (VAChT KD(HOM)) leads to decreased cholinergic tone, autonomic imbalance and a phenotype resembling cardiac dysfunction. In order to further understand the molecular changes resulting from chronic long-term decrease in parasympathetic tone, we undertook a transcriptome-based, microarray-driven approach to analyze gene expression changes in ventricular tissue from VAChT KD(HOM) mice. We demonstrate that a decrease in cholinergic tone is associated with alterations in gene expression in mutant hearts, which might contribute to increased ROS levels observed in these cardiomyocytes. In contrast, in another model of cardiac remodeling and autonomic imbalance, induced through chronic isoproterenol treatment to increase sympathetic drive, these genes did not appear to be altered in a pattern similar to that observed in VAChT KD(HOM) hearts. These data suggest the importance of maintaining a fine balance between the two branches of the autonomic nervous system and the significance of absolute levels of cholinergic tone in proper cardiac function.

Mechanical tension increases CCN2/CTGF expression and proliferation in gingival fibroblasts via a TGFß-dependent mechanism.

Unlike skin, oral gingival do not scar in response to tissue injury. Fibroblasts, the cell type responsible for connective tissue repair and scarring, are exposed to mechanical tension during normal and pathological conditions including wound healing and fibrogenesis. Understanding how human gingival fibroblasts respond to mechanical tension is likely to yield valuable insights not only into gingival function but also into the molecular basis of scarless repair. CCN2/connective tissue growth factor is potently induced in fibroblasts during tissue repair and fibrogenesis. We subjected gingival fibroblasts to cyclical strain (up to 72 hours) using the Flexercell system and showed that CCN2 mRNA and protein was induced by strain. Strain caused the rapid activation of latent TGFß, in a fashion that was reduced by blebbistatin and FAK/src inhibition, and the induction of endothelin (ET-1) mRNA and protein expression. Strain did not cause induction of α-smooth muscle actin or collagen type I mRNAs (proteins promoting scarring); but induced a cohort of pro-proliferative mRNAs and cell proliferation. Compared to dermal fibroblasts, gingival fibroblasts showed reduced ability to respond to TGFß by inducing fibrogenic mRNAs; addition of ET-1 rescued this phenotype. Pharmacological inhibition of the TGFß type I (ALK5) receptor, the endothelin A/B receptors and FAK/src significantly reduced the induction of CCN2 and pro-proliferative mRNAs and cell proliferation. Controlling TGFß, ET-1 and FAK/src activity may be useful in controlling responses to mechanical strain in the gingiva and may be of value in controlling fibroproliferative conditions such as gingival hyperplasia; controlling ET-1 may be of benefit in controlling scarring in response to injury in the skin.

Gingival fibroblasts display reduced adhesion and spreading on extracellular matrix: a possible basis for scarless tissue repair?

Unlike skin, oral gingiva do not scar in response to injury. The basis of this difference is likely to be revealed by comparing the responses of dermal and gingival fibroblasts to fibrogenic stimuli. Previously, we showed that, compared to dermal fibroblasts, gingival fibroblasts are less responsive to the potent pro-fibrotic cytokine TGFß, due to a reduced production of endothelin-1 (ET-1). In this report, we show that, compared to dermal fibroblasts, human gingival fibroblasts show reduced expression of pro-adhesive mRNAs and proteins including integrins α2 and α4 and focal adhesion kinase (FAK). Consistent with these observations, gingival fibroblasts are less able to adhere to and spread on both fibronectin and type I collagen. Moreover, the enhanced production of ET-1 mRNA and protein in dermal fibroblasts is reduced by the FAK/src inhibitor PP2. Given our previous observations suggesting that fibrotic fibroblasts display elevated adhesive properties, our data suggest that scarring potential may be based, at least in part, on differences in adhesive properties among fibroblasts resident in connective tissue. Controlling adhesive properties may be of benefit in controlling scarring in response to tissue injury.