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NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors in the innate immune system. In particular, the TLR4-mediated immune response has been implicated in ischemia-induced tissue injury. Mounting evidence supports a detrimental role of the innate immune system in the pathophysiology of skeletal muscle damage in patients with chronic limb-threatening ischemia (CLTI), in whom patient-oriented functional outcomes are poor. The overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction and damage in CLTI., Methods: The role of TLR4 in ischemic muscle was investigated by (1) studying TLR4 expression and distribution in human gastrocnemius muscle biopsies, (2) evaluating the functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) assessing the therapeutic potential of modulating TLR4 signaling in ischemic muscle in a mouse hindlimb ischemia model., Results: TLR4 was found to be expressed in human muscle biopsies, with significant upregulation in samples from patients with CLTI. In vitro studies using cultured human myotubes demonstrated upregulation of TLR4 in ischemia, with activation of the downstream signaling pathway. Inhibition of TLR4 before ischemia was associated with reduced ischemia-induced apoptosis. Upregulation of TLR4 also occurred in ischemia in vivo and TLR4 inhibition was associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb., Conclusions: TLR4 is upregulated and activated in ischemic skeletal muscle in patients with CLTI. Modulating TLR4 signaling in vitro and in vivo was associated with attenuation of ischemia-induced skeletal muscle damage. This strategy could be explored further for potential clinical application., Competing Interests: None., (© 2024 by the Society for Vascular Surgery. Published by Elsevier Inc.)

Microplastics (MPs) have attracted widespread attention as an emerging environmental pollutant. Especially in aquatic ecosystems, the harm of MPs to aquatic animals has increasingly become a severe environmental problem. In this study, we constructed a carp polystyrene microplastics (PS-MPs) exposure model to explore the damage and mechanism of PS-MPs exposure to carp myocardial tissue. The results of H&E, TUNEL, and AO/EB staining showed that PS-MPs exposure could induce inflammation, apoptosis, and necrosis in carp myocardial tissue and cardiomyocytes. In addition, our study explored the targeting relationship between PS-MPs and TLR4 and found that PS-MPs exposure could significantly increase the expression of TLR4 pathway-related factors. As the concentration of PS-MPs increased, the NF-κB pathway and inflammation-related factors increased dose-dependent. In addition, myocardial injury induced by exposure to PS-MPs was predominantly apoptotic, accompanied by necrosis. In short, our data suggest that PS-MPs cause damage to myocardial tissue via the TLR4\NF-κB pathway. The above findings enrich the theory of toxicological studies on PS-MPs and provide an essential reference for aquaculture., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Recent evidence has indicated that alterations in energy metabolism play a critical role in the pathogenesis of fibrotic diseases. Studies have suggested that 'metabolic reprogramming' involving the glycolysis and oxidative phosphorylation (OXPHOS) in cells lead to an enhanced generation of energy and biosynthesis. The aim of this study was to assess the molecular basis of changes in fibrotic metabolism in systemic sclerosis (Scleroderma; SSc) and highlight the most appropriate targets for anti-fibrotic therapies., Materials and Methods: Dermal fibroblasts were isolated from five SSc patients and five healthy donors. Cells were cultured in medium with/without TGF-β1 and with/without ALK5, pan-PIM or ATM kinase inhibitors. Extracellular flux analyses were performed to evaluate glycolytic and mitochondrial respiratory function. The mitochondrial network in TMRM-stained cells was visualized by confocal laser-scanning microscopy, followed by semi-automatic analysis on the ImageJ platform. Protein expression of ECM and fibroblast components, glycolytic enzymes, subunits of the five OXPHOS complexes, and dynamin-related GTPases and receptors involved in mitochondrial fission/fusion were assessed by western blotting., Results: Enhanced mitochondrial respiration coupled to ATP production was observed in SSc fibroblasts at the expense of spare respiratory capacity. Although no difference was found in glycolysis when comparing SSc with healthy control fibroblasts, levels of phophofructokinase-1 isoform PFKM were significantly lower in SSc fibroblasts ( P <0.05). Our results suggest that the number of respirasomes is decreased in the SSc mitochondria; however, the organelles formed a hyperfused network, which is thought to increase mitochondrial ATP production through complementation. The increased mitochondrial fusion correlated with a change in expression levels of regulators of mitochondrial morphology, including decreased levels of DRP1, increased levels of MIEF2 and changes in OPA1 isoform ratios. TGF-β1 treatment strongly stimulated glycolysis and mitochondrial respiration and induced the expression of fibrotic markers. The pan-PIM kinase inhibitor had no effect, whereas both ALK5 and ATM kinase inhibition abrogated TGF-β1-mediated fibroblast activation, and upregulation of glycolysis and respiration., Conclusions: Our data provide evidence for a novel mechanism(s) by which SSc fibroblasts exhibit altered metabolic programs and highlight changes in respiration and dysregulated mitochondrial morphology and function, which can be selectively targeted by small molecule kinase inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cantanhede, Liu, Liu, Balbuena Rodriguez, Shiwen, Ong, Denton, Ponticos, Xiong, Lima-Filho, Abraham, Abu-Hanna and Taanman.)

TARP syndrome is a rare X-linked genetic condition caused by mutations in the RBM10 gene. Primary clinical characteristics of TARP syndrome include Talipes equinovarus, Atrial septal defect, Robin sequence and Persistent left superior vena cava. Newly reported cases identified a few novel RBM10 variants and atypical manifestations associated with TARP syndrome, thus expanding the genetic and clinical spectrum of TARP syndrome. Here we report a molecularly confirmed TARP syndrome with distinctive clinical features including pulmonary arteriovenous malformation, single umbilical artery, and coagulopathy. We identified a frameshift RBM10 variant that might be associated with his distinctive clinical features., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Daicheng, Shiwen, Jingxuan, Junbo and Bo.)

Environmental pollutant cadmium (Cd) can cause macrophage dysfunction, and the imbalance of M1/M2 is involved in the process of tissue fibrosis. In order to explore the effect of subacute CdCl 2 exposure on pig lung tissue fibers and its mechanism, based on the establishment of this model, ICP-MS, H&E staining, Masson staining, Immunofluorescence, RT-PCR, and Western Blot methods were used to detect related indicators. The results found that lung tissue fibrosis, Cd content significantly increased, lung tissue ion disturbance, miR-20a-3p down-regulation, M1/M2 imbalance, LXA4/FPR2 content decreased, MDA content increased, NF-κB/NLRP3, TGFβ pathway, PPARγ/Wnt pathway activated, and the expression of fibrosis-related factors increased. The above results indicate that subacute CdCl 2 exposure increase Cd content in the pig lungs, which leads to M1/M2 imbalance and down-regulates the content of LXA4/FPR2, further activates the oxidative stress/NF-κB/NLRP3 pathway, thereby activating the TGFβ and PPARγ/Wnt pathways to induce fibrosis. This study aims to reveal the toxic effects of CdCl 2 and will provide new insights into the toxicology of Cd., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The TβRII∆k-fib transgenic (TG) mouse model of scleroderma replicates key fibrotic and vasculopathic complications of systemic sclerosis through fibroblast-directed upregulation of TGFβ signalling. We have examined peroxisome proliferator-activated receptor (PPAR) pathway perturbation in this model and explored the impact of the pan-PPAR agonist lanifibranor on the cardiorespiratory phenotype., Methods: PPAR pathway gene and protein expression differences from TG and WT sex-matched littermate mice were determined at baseline and following administration of one of two doses of lanifibranor (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. The prevention of bleomycin-induced lung fibrosis and SU5416-induced pulmonary hypertension by lanifibranor was explored., Results: Gene expression data were consistent with the downregulation of the PPAR pathway in the TβRII∆k-fib mouse model. TG mice treated with high-dose lanifibranor demonstrated significant protection from lung fibrosis after bleomycin and from right ventricular hypertrophy following induction of pulmonary hypertension by SU5416, despite no significant change in right ventricular systolic pressure., Conclusions: In the TβRII∆k-fib mouse strain, treatment with 100 mg/kg lanifibranor reduces the development of lung fibrosis and right ventricular hypertrophy induced by bleomycin or SU5416, respectively. Reduced PPAR activity may contribute to the exaggerated fibroproliferative response to tissue injury in this transgenic model of scleroderma and its pulmonary complications., (© 2021. The Author(s).)

The environmental problem of Microplastics (MPs) pollution poses a great threat to human and animal health, which has attracted global attention. The physiological integrity of skeletal muscle is extremely important for the survival of animals. Here, we investigated the effect of two size polystyrene microplastics (PS-MPs, 1-10 µm and 50-100 µm) on the growth of anterior tibial (TA) muscle and repair after injury in mice. Results showed that the regeneration of skeletal muscle was delayed by PS-MPs exposure and was negatively correlated with particle size. H&E staining and Oil red O staining showed that PS-MPs exposure reduced the average cross-sectional area (CSA) and diameter of the muscle fibers, increased lipid deposition. Further mechanistic research displayed that though PS-MPs treatment did not affect cell viability of myoblast, it aggravated intracellular ROS generation and oxidative stress, inhibited myogenic differentiation by decreasing the phosphorylation of p38 MAPK, and promote adipogenic differentiation by increasing the expression of NF-κB, which could be alleviated by NAC. In brief, our data demonstrated that the ROS overproduction caused by PS-MPs disturbed the regeneration of skeletal muscle and directed the fate of satellite cells in mice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recent studies identified a novel programmed and regulated cell death that was characterized by a necrotic cell death morphology, termed necroptosis. Lead (Pb) is known as a persistent inorganic environmental pollutant that affects the health of humans and animals worldwide. However, there are no detailed reports of Pb-induced necroptosis of immune tissue. Selenium (Se) is a trace element that antagonizes the toxicity of heavy metals. Here, chickens were randomly divided into four groups, treated with Pb ((CH 3 OO) 2 Pb, 150 mg/kg) and/or Se (Na 2 SeO 3 , 2 mg/kg), aim to study the effect and mechanism of necroptosis in Pb-induced spleen injury and the antagonistic effects of Se on Pb toxicity. Our results showed that Pb exposure evidently increased the accumulation of Pb in spleen and caused necroptosis by upregulating the expression of RIP1, RIP3 and MLKL, and decreasing Caspase8 expression. Meanwhile, Pb treatment inhibited the activities of SOD, GPX, and CAT, caused the accumulation of NO and MDA, and induced oxidative stress, which promoted the expression of MAPK/NF-κB pathway genes (ERK, JNK, P38, NF-κB and TNF-α) and activated HSPs (HSP27, HSP40, HSP60, HSP70 and HSP90). However, the increased content of Pb in spleen and Pb-caused necroptosis were inhibited by Se cotreatment. Overall, we conclude that Se can prevent Pb-induced necroptosis by restoring antioxidant functions and blocking the MAPK/NF-κB pathway and HSPs activation in chicken spleen., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Critical limb ischemia (CLI) is associated with skeletal muscle damage. However, the pathophysiology of the muscle damage is poorly understood. Toll-like receptors (TLR) have been attributed to play a role in ischemia-induced tissue damage but their role in skeletal muscle damage in CLI is unknown. TLR2 and TLR6 expression was found to be upregulated in skeletal muscle of patients with CLI. In vitro, ischemia led to upregulation of TLR2 and TLR6 by myotubes, and activation of the downstream TLR signaling pathway. Ischemia-induced activation of the TLR signaling pathway led to secretion of the pro-inflammatory cytokine interleukin-6 and muscle apoptosis, which were abrogated by neutralising TLR2 and TLR6 antibodies. Our study demonstrates that TLR2 and TLR6 are upregulated in ischemic muscle and play a role in ischemia-induced muscle damage. Thus, manipulating the TLR pathway locally may be of potential therapeutic benefit.

Plant lectins are carbohydrate-binding proteins, which can interact with cell surfaces to initiate anti-inflammatory pathways, as well as immunomodulatory functions. Here, we have extracted, purified and part-characterized the bioactivity of Jacalin, Frutalin, DAL and PNA, before evaluating their potential for wound healing in cultured human skin fibroblasts. Only Frutalin stimulated fibroblast migration in vitro, prompting further studies which established its low cytotoxicity and interaction with TLR4 receptors. Frutalin also increased p-ERK expression and stimulated IL-6 secretion. The in vivo potential of Frutalin for wound healing was then assessed in hybrid combination with the polysaccharide galactomannan, purified from Caesalpinia pulcherrima seeds, using both hydrogel and membrane scaffolds formulations. Physical-chemical characterization of the hybrid showed that lectin-galactomannan interactions increased the pseudoplastic behaviour of solutions, reducing viscosity and increasing Frutalin's concentration. Furthermore, infrared spectroscopy revealed -OH band displacement, likely caused by interaction of Frutalin with galactose residues present on galactomannan chains, while average membrane porosity was 100 μm, sufficient to ensure water vapor permeability. Accelerated angiogenesis and increased fibroblast and keratinocyte proliferation were observed with the optimal hybrid recovering the lesioned area after 11 days. Our findings indicate Frutalin as a biomolecule with potential for tissue repair, regeneration and chronic wound healing., (Copyright © 2018. Published by Elsevier B.V.)

Background: Present investigation evaluates the beneficial effect of fangchinoline on cerebral ischemia induced neuronal degeneration in neonatal rats and also postulates the possible mechanism of its action., Methodology: Cerebral ischemia was produced by the ligation of right common carotid artery in neonatal rats on postnatal day 5 (P5) and further pups were treated with fangchinoline 3, 10 and 30 mg/kg, i.p. for the period of 3 days. Effect of fangchinoline was estimated by determining the brain injury and enzyme linked immunosorbent assay (ELISA) method was used for the estimation of pro-inflammatory mediators and markers of oxidative stress in the cerebral tissues of neonatal rats. Moreover western blot assay and histopathology study was also performed on the brain tissue., Results: Result of this investigation reveals that the percentage of brain injury significantly reduces and enhancement of myelin basic protein in the cerebral tissues of fangchinoline than ischemic group. Treatment with fangchinoline attenuates the altered level of proinflammatory mediators and markers of oxidative stress in the cerebral tissue of cerebral ischemia induced neuronal injury neonatal rats. Moreover expressions of inducible nitric oxide synthtase (iNOS), vascular endothelial growth factor (VEGF), p53 and nuclear receptor factor-2 (Nrf2) in the brain tissue attenuated by fangchinoline treated group., Conclusion: In conclusion, fangchinoline ameliorates the cerebral ischemia induced neuronal injury in neonatal rats by enhancing angiogenesis molecules., Competing Interests: Conflict of interest No

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4., (© 2016 The Author(s).)

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.

Introduction: Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients., Methods: Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors., Results: Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator., Conclusions: Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.

Skin involvement with dermal fibrosis is a hallmark of systemic sclerosis (SSc), and keratinocytes may be critical regulators of fibroblast function through secretion of chemo-attracting agents, as well as through growth factors and cytokines influencing the phenotype and proliferation rate of fibroblasts. Epithelial-fibroblast interactions have an important role in fibrosis in general. We have characterized the SSc epidermis and asked whether SSc-injured epidermal cells release factors capable of promoting fibrosis. Our results show that the SSc epidermis is hypertrophic, and has altered expression of terminal differentiation markers involucrin, loricrin, and filaggrin. Multiplex profiling revealed that SSc epidermal explants release increased levels of CCN2 and S100A9. CCN2 induction was found to spread into the upper papillary dermis, whereas S100A9 was shown to induce fibroblast proliferation and to enhance fibroblast CCN2 expression via Toll-like receptor 4. These data suggest that the SSc epidermis provides an important source of pro-fibrotic CCN2 and proinflammatory S100A9 in SSc skin, and therefore contributes to the fibrosis and inflammation seen in the disease.

Objective: Managing critical limb ischemia (CLI) is challenging. Furthermore, ischemic myopathy prevents good functional outcome after revascularization. Hence, we have focused on limiting the tissue damage rather than angiogenesis, which has traditionally been the motivation to develop nonsurgical treatments for CLI. Erythropoietin (EPO) protects ischemic tissue, and this property may also benefit CLI. The objective of this study was to examine the expression of the tissue-protective EPO receptor complex (EPOR-CD131 [β-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor]) in skeletal muscle obtained from humans with CLI. Because native EPO is thrombogenic, the antiapoptotic and anti-inflammatory effects of a nonhematopoietic helix-B peptide of EPO (ARA 290) were investigated on ischemic myotubes in vitro., Methods: Tissue was obtained from gastrocnemius muscle of 12 patients undergoing amputation for CLI and from 12 patients without limb ischemia. The expression of EPOR and CD131 was demonstrated by immunohistochemistry and Western blot. A validated in vitro model of myotube ischemia was used in which mature C2C12 myotubes were cultured 6 to 12 hours in a depleted media and gas mixture (20% CO2 and 80% N2). The myotubes were pretreated with EPO or ARA 290 before exposure to simulated ischemia. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase release assay. Enzyme-linked immunosorbent assay measured the inflammatory cytokines., Results: EPOR and CD131 were expressed and significantly upregulated in CLI (average optical density [OD] in Western blot [control vs CLI] EPOR, 0.05 U vs 0.1 U; CD131, 0.10 U vs 0.22 U; P < .01). There was colocalization of EPOR and CD131 in the sarcolemma (cell membrane) of the skeletal myofiber. There was no difference in the distribution of colocalization between the CLI and the normal muscle. The ischemic myotubes treated by ARA 290 in vitro had a significantly decreased number of apoptotic cells (ischemia vs ischemia plus ARA 290: 71.1% vs 55.1%; P < .01), cleaved caspase-3 (OD of ischemia vs ischemia plus ARA 290: 0.15 U vs 0.02 U; P < .01), lactate dehydrogenase release (ischemia vs ischemia plus ARA 290: 32.5 U/L vs 21.3 U/L; P < .01), and IL-6 release (OD at 450 nm, ischemia vs ischemia plus ARA 290: 0.18 vs 0.13; P < .01)., Conclusions: This study demonstrates the expression and the upregulation of EPOR and CD131 in CLI and also shows that EPOR and CDI are colocalized in the cell membrane of both ischemic and control muscle fiber. The in vitro experiments demonstrate that ARA 290 decreases inflammation and apoptosis of ischemic myotubes. ARA 290 may potentially be used as adjunctive treatment for CLI., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Endothelin 1 (ET-1) is overexpressed in cancer, contributing to disease progression. We previously showed that ET-1 stimulated proliferative, migratory, and contractile tumorigenic effects via the ET(A) receptor. Here, for the first time, we evaluate zibotentan, a specific ET(A) receptor antagonist, in the setting of colorectal cancer, in cellular models. Pharmacologic characteristics were further determined in patient tissues. Colorectal cancer lines (n = 4) and fibroblast strains (n = 6), isolated from uninvolved areas of colorectal cancer specimens, were exposed to ET-1 and/or ET(A)/(B) receptor antagonists. Proliferation (methylene blue), migration (scratch wounds), and contraction (gel lattices) were assessed. Receptor distribution and binding characteristics (K(d), B(max)) were determined using autoradiography on tissue sections and homogenates and cytospun cells, supported by immunohistochemistry. Proliferation was inhibited by ET(A) (zibotentan > BQ123; P < 0.05), migration by ET(B) > ET(A), and contraction by combined ET(A) and ET(B) antagonism. Intense ET-1 stromal binding correlated with fibroblasts and endothelial cells. Colorectal cancer lines and fibroblasts revealed high density and affinity ET-1 binding (B(max) = 2.435 fmol/1 × 10(6) cells, K(d) = 367.7 pmol/L; B(max) = 3.03 fmol/1 × 10(6) cells, K(d) = 213.6 pmol/L). In cancer tissues, ET(A) receptor antagonists (zibotentan; BQ123) reduced ET-1 binding more effectively (IC(50): 0.1-10 μmol/L) than ET(B) receptor antagonist BQ788 (∼IC(50), 1 mmol/L). ET-1 stimulated cancer-contributory processes. Its localization to tumor stroma, with greatest binding/affinity to fibroblasts, implicates these cells in tumor progression. ET(A) receptor upregulation in cancer tissues and its role in tumorigenic processes show the receptor's importance in therapeutic targeting. Zibotentan, the most specific ET(A) receptor antagonist available, showed the greatest inhibition of ET-1 binding. With its known safety profile, we provide evidence for zibotentan's potential role as adjuvant therapy in colorectal cancer.

Background: Platelet-derived growth factor (PDGF) signalling is essential for many key cellular processes in mesenchymal cells. As there is redundancy in signalling between the five PDGF ligand isoforms and three PDGF receptor isoforms, and deletion of either of the receptors in vivo produces an embryonic lethal phenotype, it is not know which ligand and receptor combinations mediate specific cellular functions. Fibroblasts are key mediators in wound healing and tissues repair. Recent clinical trials using broad spectrum tyrosine kinase inhibitors in fibrotic diseases have highlighted the need to further examine the specific cellular roles each of the tyrosine kinases plays in fibrotic processes. In this study, we used PDGFR-specific neutralising antibodies to dissect out receptor-specific signalling events in fibroblasts in vitro, to further understand key cellular processes involved in wound healing and tissue repair., Results: Neutralising antibodies against PDGFRs were shown to block signalling through PDGFRα and PDGFRβ receptors, reduce human PDGF-AA and PDGF-BB-induced collagen gel remodelling in dermal fibroblasts, and reduce migration stimulated by all PDGF ligands in human dermal and lung fibroblasts., Conclusions: PDGFRα and PDGFRβ neutralising antibodies can be a useful tool in studying PDGFR isoform-specific cellular events.

Erythropoietin (EPO) has tissue-protective properties, but it increases the risk of thromboembolism by raising the haemoglobin concentration. New generation of EPO derivatives is tissue protective without the haematopoietic side effects. Preclinical studies have demonstrated their effectiveness and safety. This paper summarizes the development in EPO derivatives with emphasis on their potential use in critical limb ischaemia.

In the Western world, peripheral vascular disease (PVD) has a high prevalence with high morbidity and mortality. In a large percentage of these patients, lower limb amputation is still required. Studies of ischaemic skeletal muscle disclosed evidence of endogenous angiogenesis and adaptive skeletal muscle metabolic changes in response to hypoxia. Chemokines are potent chemoattractant cytokines that regulate leukocyte trafficking in homeostatic and inflammatory processes. More than 50 different chemokines and 20 different chemokine receptors have been cloned. The chemokine stromal-cell-derived factor-1 (SDF-1 aka CXCL12) is a constitutively expressed and inducible chemokine that regulates multiple physiological processes, including embryonic development and organ homeostasis. The biologic effects of SDF-1 are mediated by chemokine receptor CXCR4, a 352 amino acid rhodopsin-like transmembrane-specific G protein-coupled receptor (GPCR). There is evidence that the administration of SDF-1 increases blood flow and perfusion via recruitment of endothelial progenitor cells (EPCs). This review will focus on the role of the SDF-1/CXCR4 system in the pathophysiology of PVD and discuss their potential as therapeutic targets for PVD.

Critical limb ischemia causes severe damage to the skeletal muscle. This study develops a reproducible model of myotube ischemia by simulating, in vitro, the critical parameters that occur in skeletal muscle ischemia. Monolayers of C2C12 myoblasts were differentiated into mature myotubes and exposed to nutrition depletion, hypoxia and hypercapnia for variable time periods. A range of culture media and gas mixture combinations were used to obtain an optimum ischemic environment. Nuclear staining, cleaved caspase-3 and lactate dehydrogenase (LDH) release assay were used to assess apoptosis and myotube survival. HIF-1α concentration of cell lysates, pH of conditioned media as well as partial pressures of oxygen (PO₂) and carbon dioxide (PCO₂) in the media were used to confirm ischemic simulation. Culturing myotubes in depleted media, in a gas mixture containing 20% CO+80% N₂ for 6-12 h increased the PCO₂ and decreased the pH and PO₂ of culture media. This attempts to mimic the in vivo ischemic state of skeletal muscle. These conditions were used to study the potential tissue-protective effects of erythropoietin (EPO) in C2C12 myotubes exposed to ischemia. EPO (60 ng/ml) suppressed LDH release, decreased cleaved caspase-3 and reduced the number of apoptotic nuclei, suggesting significantly decreased ischemia-induced apoptosis in myotubes (P<0.01) and a potential role in tissue protection. Additional therapeutic agents designed for tissue protection can also be evaluated using this model.

Background: Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations. Vasculopathy is an early feature of the disease which leads to a chronic hypoxic environment in the tissues. Paradoxically, there is a lack of angiogenesis. We hypothesised that this may in part be due to a nonphysiological, overriding upregulation in antiangiogenic factors produced by the hypoxic tissues. We considered thrombospondin 1 (TSP-1) as a candidate antiangiogenic factor., Results: Conditioned media from human microvascular endothelial cells cultured in both normoxic and hypoxic environments were able to block endothelial cell proliferation, with the latter environment having a more profound effect. Filtration to remove > 100-kDa proteins or heparin-binding proteins from the conditioned media eliminated their antiproliferative effect. TSP-1 was expressed in high concentrations in the hypoxic media, as was vascular endothelial growth factor (VEGF). Depletion of TSP-1 from the media by immunoprecipitation reduced the antiproliferative effect. We then show that, in a dose-dependent fashion, recombinant TSP-1 blocks the proliferation of endothelial cells. Immunohistochemistry of skin biopsy material revealed that TSP-1 expression was significantly higher throughout the skin of patients with SSc compared with healthy controls., Conclusions: Despite the environment of chronic tissue hypoxia in SSc, there is a paradoxical absence of angiogenesis. This is thought to be due in part to aberrant expression of antiangiogenic factors, including TSP-1. We have demonstrated that TSP-1 is released in high concentrations by hypoxic endothelial cells. The conditioned media from these cells is able to block proliferation and induce apoptosis in microvascular endothelial cells, an effect that is reduced when TSP-1 is immunoprecipitated out. Further, we have shown that recombinant TSP-1 is able to block proliferation and induce apoptosis at concentrations consistent with those found in the plasma of patients with SSc and that its effect occurs in the presence of elevated VEGF levels. Taken together, these data are consistent with a model wherein injured microvascular cells in SSc fail to repair because of dysregulated induction of TSP-1 in the hypoxic tissues.

Rationale: Fibrotic response to lung injury depends on development of a fibrogenic population of myofibroblasts. The importance of resident interstitial fibroblasts and role of transforming growth factor β (TGFβ) in this process is unclear., Objectives: To define the importance of TGFβ signaling in resident lung fibroblasts in the development of experimental pulmonary fibrosis., Methods: A compound genetic strategy in which mice homozygous for a floxed high-affinity type II TGFβ receptor (TβRII) allele were crossed with a transgenic strain harboring a fibroblast-specific transgene encoding ligand-dependent Cre-recombinase was used. TβRII was deleted by postnatal administration of tamoxifen over 5 days to compound mutant mice with appropriate littermate controls. Illumina microarray gene profiling and quantitative reverse transcriptase-polymerase chain reaction were used to confirm anergy to TGFβ in explanted lung fibroblasts. Bleomycin lung injury was used to induce lung fibrosis, which was analyzed by histology and biochemical methods. Immunofluorescence was used to define cell populations after lung injury., Measurements and Main Results: There was significant attenuation of fibrosis in mice after deletion of TβRII in resident fibroblasts. At 7 days after injury the number of fibrocytes and myofibroblasts was substantially reduced. Potential regulators of fibrosis were suggested by gene expression profiles that identified key candidate profibrotic genes, including connective tissue growth factor and endothelin-1 expressed by wild-type but not mutant lung fibroblasts., Conclusions: Intact TGFβ signaling in resident pulmonary fibroblasts is essential for pulmonary fibrosis to develop. Our data support a key regulatory role of these cells in determining fibrocyte recruitment and myofibroblast differentiation.

Systemic sclerosis (SSc) is a disorder of systemic and dermal fibrosis of uncertain etiology. Recently, we found that SSc epidermis is abnormal, taking on an activated phenotype observed during wound healing and tissue repair. As epithelial-fibroblast interactions are important during wound repair and in fibrosis in general, we investigated further the phenotype of the SSc epidermis, and tested whether the SSc epidermis provides a pro-fibrotic stimulus to fibroblasts. In this study we show that in SSc epidermis keratinocyte maturation is delayed, and wound-associated keratins 6 and 16 are induced, in both involved and clinically uninvolved skin. Phosphorylation array analysis revealed induction of stress-induced mitogen-activated protein kinase signaling and mesenchymal feedback through hepatocyte growth factor/c-Met in SSc epidermis. SSc epidermal cells maintained with normal fibroblasts in three-dimensional co-culture were found to stimulate fibroblasts, leading to contractility and connective tissue growth factor expression. These effects depend on elevation of IL-1alpha by the epidermal cells and induction of endothelin-1 and transforming growth factor-beta in fibroblasts. Antagonism of endogenous IL-1alpha using IL-1 receptor antagonist blocked gel contraction by SSc epidermis. We propose that in SSc, epidermal cells are in a persistently activated state and are able to promote dermal fibrosis. These findings are important because biologic therapies could target epithelial-fibroblast interactions in the disease.

Fibroblasts release prostaglandins and express a range of prostanoid receptors. However the importance of prostaglandins in fibroblast biology have not been fully explored. Our studies showed that the prostaglandin metabolite PGI(2) blocks the activation of fibroblasts, antagonising the induction of Ras/MEK/ERK signalling by TGFbeta. Endogenous PGI(2) acts so as to limit the activation of fibroblasts following tissue injury. By contrast PGE(2) induced in injured tissues or disease states may promote recruitment of inflammatory cells and lead to secondary activation of fibroblasts. The effects of PGI(2) on cell signaling could be manipulated to inhibit fibrosis in patients.

Aim: The safety and potential efficacy of a chimaeric anti-tumour necrosis factor alpha monoclonal antibody (infliximab) were examined in diffuse cutaneous systemic sclerosis (dcSSc)., Methods: A 26-week open-label pilot study in which 16 cases of dcSSc received five infusions of infliximab (5 mg/kg). Clinical assessment included skin sclerosis score, scleroderma health assessment questionnaire, self-reported functional score and physician global visual analogue scale. Collagen turnover, skin biopsy analysis and full safety evaluation were performed., Results: There was no significant change in skin score at 26 weeks but a trend for lower modified Rodnan skin score at 22 weeks (OR 17, 95% CI 6 to 46) compared with peak value (OR 29, 95% CI 11 to 44; p = 0.10). Serum aminoterminal propeptide of type III collagen level was significantly lower at week 26 compared with baseline (p = 0.03). Secretion of type I collagen by dermal fibroblasts was reduced at 26 weeks compared with baseline (p = 0.02). There were no deaths during the study and no suspected unexpected serious adverse reactions. 21 serious adverse events (AE) occurred in seven subjects, mostly attributable to dcSSc. 127 distinct AE occurred in 16 subjects. Of these, 19 AE (15%) were probably or definitely related to infliximab treatment. Eight (50%) patients prematurely discontinued infliximab. Anti-infliximab antibodies developed during the study in five subjects and were significantly associated with suspected infusion reactions (p = 0.025)., Conclusion: In dcSSc infliximab did not show clear benefit at 26 weeks but was associated with clinical stabilisation and a fall in two laboratory markers of collagen synthesis. The frequency of suspected infusion reactions may warrant additional immunosuppression in any future studies in systemic sclerosis.

The coupled antigen, avermectin (AVM) and bovine serum albumin (BSA), was artificially synthesized. Female 8-week-old BALB/c mice were immunized with AVM-BSA emulsified in Freund's adjuvant. Two monoclonal antibodies (MAbs) against AVM, named 2F2 and 2H10, were prepared by lymphocyte hybridoma technique. Ascitic fluid titers of the MAbs 2F2 and 2H10 were, respectively, 1:6400 and 1:25,600, and their subtypes were all IgMkappa. In competitive indirect ELISA (Ci-ELISA), 50% inhibiting concentration (IC(50)) of the MAb 2H10 for avermectin was 101 ng/mL. In specific assay, the MAb 2H10 could not react with ivermectin, doramectin, erythromycin, and albomycin. These data demonstrated that the MAb 2H10 will have a potential use for developing diagnostic reagents of avermectin residues.

Scleroderma [systemic sclerosis (SSc)] is a spectrum of connective tissue diseases characterized by micro- and macro-vasculopathy, inflammation and autoimmunity and tissue remodelling that often leads to excessive scarring and fibrosis in both interstitial and vascular compartments. Pre-clinical investigations and gene association studies have led to improved understanding of the cell and molecular mechanisms underlying disease pathogenesis and to the identification of key molecular candidates that may represent potentially useful disease biomarkers and effective therapeutic targets. Studies on the endothelin (ET) system, pre-dominantly ET-1 and the cell surface receptors [type A (ET(A))] and type B (ET(B))], have provided evidence for an important role of this system in the vascular and fibrotic pathologies in SSc. To date, promising clinical results, utilizing dual/mixed ET receptor antagonism have been obtained in two of the vascular complications associated with SSc, ischaemic digital ulceration and pulmonary arterial hypertension. Evidence suggests that ET-1 is able to activate and re-program the functional phenotypes of vascular smooth muscle cells, microvascular pericytes and tissue fibroblasts into pro-fibrogenic cell populations with myofibroblasts-like properties. The impact of receptor-selective, over mixed-receptor, antagonism has also been studied in vitro with respect to cell differentiation and proliferation, extracellular matrix synthesis, production and deposition and in pathological cellular contraction. However, the complexity of the ET system, potential for receptor cross-talk, interactions with down-stream signal transduction cascades, as well as the potent inter-relationships with other important ligand-receptor pathways have made in vivo studies difficult to unravel. Moreover, little information is available on the role of the ET system and receptor selectivity in the recruitment and activation of mesenchymal progenitor cells in tissue remodelling and fibrosis or on the early inflammatory response. Here, we discuss the available pre-clinical evidence for the role of the ET system in tissue repair, scarring and fibrosis, using the connective tissue diseases SSc and model systems of fibrogenesis.

By providing a source of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts, microvascular pericytes contribute to the matrix remodeling that occurs during tissue repair. However, the extent to which pericytes may contribute to the fibroblast phenotype post-repair is unknown. In this report, we test whether pericytes isolated from human placenta can in principle become fibroblast-like. Pericytes were cultured in vitro for 11 passages. The Affymetrix mRNA expression profile of passage 2 and passage 11 pericytes was compared. The expression of type I collagen, thrombospondin and fibronectin mRNAs was induced by passaging pericytes in culture. This induction of a fibroblast phenotype was paralleled by induction of connective tissue growth factor (CTGF/CCN2) and type I collagen protein expression and the fibroblast marker ASO2. These results indicate that, in principle, pericytes have the capacity to become fibroblast-like and that pericytes may contribute to the population of fibroblasts in a healed wound.

Previous attempts to delete type II TGFbeta receptor (TbetaRII) in fibroblasts have precluded examination of adult mice due to early mortality. We have selectively deleted TbetaRII postnatally in differentiated connective tissue fibroblasts using an inducible Cre-Lox strategy. Tamoxifen-dependent Cre recombinase linked to a fibroblast-specific regulatory sequence from the proalpha2(I)collagen gene permitted deletion of floxed TbetaRII alleles. After postnatal deletion of TbetaRII in fibroblasts, healing of excisional skin wounds in adults showed markedly attenuated dermal scar formation, defective wound contraction and enhanced epidermal proliferation. These findings support a pivotal role for transforming growth factor beta (TGFbeta) signalling in fibroblasts in regulating normal skin wound healing. Explanted dermal fibroblasts from TbetaRII-null-fib mice showed impaired migration and did not generate normal contractile biomechanical forces in fixed collagen gels nor develop alpha-smooth muscle antigen-rich stress fibers in response to TGFbeta1. Surprisingly, some TGFbeta-regulated proteins, including connective tissue growth factor (CTGF), were basally upregulated in TbetaRII-null fibroblasts and this was dependent on extracellular signal-regulated kinase 1/2 activity in these cells. This suggests that other intracellular pathways regulating CTGF expression may partially compensate for disruption of TGFbeta signalling in fibroblasts. Together, our data confirm that expression of TbetaRII in differentiated dermal fibroblasts is essential for normal wound healing and demonstrate a critical role in the development and function of myofibroblasts.

Objective: To identify using proteomic analysis, proteins of altered abundance in the skin of patients with SSc., Methods: 4 mm excision biopsies were obtained from the forearm involved skin of 12 diffuse SSc patients and 12 healthy controls. Two-dimensional gel electrophoresis was used to separate and define proteins in normal and SSc skin biopsy material. Proteins of altered abundance in the disease were formally identified by mass spectroscopy. Abnormalities of the epidermis were confirmed by immunohistochemistry., Results: Proteomic analysis revealed altered abundance of proteins involved in extracellular matrix production, myofibroblast contractility, energy metabolism and response to oxidative stress. In addition, proteins specific to the epidermis and involved in epidermal cell differentiation were altered in abundance in the disease. SSc epidermis is thickened, has an expanded nucleated cell layer, and exhibits abnormal persistence of basal marker keratin 14, delayed expression of maturation markers keratin 1/10 and the induction of keratins 6 and 16, normally absent from interfollicular skin and induced following epidermal injury. These changes closely resemble the activated phenotype seen during wound healing., Conclusions: Consistent with previous models of SSc pathogenesis these data are showing increased contractility, increased extracellular matrix and response to oxidative stress in the involved skin of recent onset SSc patients. In addition, we show that SSc epidermis has an activated, wound healing phenotype. These findings are important because epidermal cells activated by injury induce and regulate local fibroblasts during wound repair.

Objective: To explore increased susceptibility to fibrosis following experimental injury to alveolar epithelial cells (AECs) in a novel transgenic mouse model of scleroderma with fibroblast-specific perturbation of transforming growth factor beta (TGFbeta) signaling (TbetaRIIDeltak-fib mice)., Methods: Wild-type (WT) and transgenic mice were injured with intratracheally administered saline or bleomycin, and the lungs were harvested for biochemical, histologic, and electron microscopic analysis., Results: Electron microscopy revealed AEC abnormalities in the lungs of untreated transgenic mice and bleomycin-treated WT mice; the lungs of transgenic mice treated with bleomycin showed severe epithelial damage. Compared with lungs from bleomycin-treated WT mice, lungs from bleomycin-treated transgenic mice demonstrated increased fibroproliferation, myofibroblast persistence, and impaired hyperplasia and increased apoptosis of type II AECs. The lungs from saline-treated transgenic mice and those from bleomycin-treated WT mice had phenotypic similarities, suggesting enhanced susceptibility to minor epithelial injury in the transgenic strain. The level of collagen was increased in the lungs from transgenic mice compared with that in the lungs from WT mice after treatment with either bleomycin or saline. Persistent fibrosis in bleomycin-treated transgenic mice was independent of ongoing neutrophil inflammation but was associated with impaired alveolar epithelial repair., Conclusion: These results suggest that in the context of fibroblast-specific perturbation of TGFbeta signaling, even minor epithelial injury induces significant fibrosis. The model supports a central role for TGFbeta in determining fibrosis and demonstrates that lung fibroblasts may regulate the response of AECs to injury. Our findings provide insight into likely pathogenic mechanisms in scleroderma-associated pulmonary fibrosis.

Objective: To investigate the contribution of heparan sulfate proteoglycan and Ras/MEK/ERK to the overexpression of profibrotic proteins and the enhanced contractile ability of dermal fibroblasts from patients with systemic sclerosis (SSc; scleroderma)., Methods: The effects of the MEK/ERK inhibitor U0126, the heparan sulfate side chain formation inhibitor beta-xyloside, and soluble heparin on the overexpression of profibrotic genes were compared in fibroblasts from lesional skin of patients with diffuse SSc and fibroblasts from healthy control subjects. Identified protein expressions were compared with the contractile abilities of fibroblasts while they resided within a collagen lattice. Forces generated were measured using a culture force monitor., Results: Inhibiting MEK/ERK with U0126 significantly reduced expression of a cohort of proadhesive and procontractile proteins that normally are overexpressed by scleroderma fibroblasts, including integrin alpha4 and integrin beta1. Antagonizing heparan sulfate side chain formation with beta-xyloside or the addition of soluble heparin prevented ERK activation, in addition to reducing the expression of these proadhesive/contractile proteins. Treatment with either U0126, beta-xyloside, or heparin resulted in a reduction in the overall peak contractile force generated by dermal fibroblasts. Blocking platelet-derived growth factor receptor with Gleevec (imatinib mesylate) reduced overall contractile ability and the elevated syndecan 4 expression and ERK activation in SSc fibroblasts., Conclusion: The results of this study suggest that heparan sulfate-dependent ERK activation contributes to the enhanced contractile ability demonstrated by dermal fibroblasts from lesional skin of patients with scleroderma. These results are consistent with the notion that the MEK/ERK procontractile pathway is dysregulated in scleroderma dermal fibroblasts. Additionally, the results suggest that antagonizing the MEK/ERK pathway is likely to modulate heparan sulfate proteoglycan activity, which in turn may have a profound effect on the fibrotic response in SSc.

Connective tissue remodeling provides mammals with a rapid mechanism to repair wounds after injury. Inappropriate activation of this reparative process leads to scarring and fibrosis. Here, we studied the effects of platelet-derived growth factor receptor-beta blockade in vivo using the platelet-derived growth factor receptor (PDGFR)-beta inhibitor imatinib mesylate on tissue repair. After 7 days, healing of wounds was delayed with significantly reduced wound closure and concomitant reduction in myofibroblast frequency, expression of fibronectin ED-A, and collagen type I. Using a collagen type I transgenic reporter mouse, we showed that inhibiting PDGFR-beta activation restricted the distribution of collagen-synthesizing cells to wound margins and dramatically reduced cell proliferation in vivo. By 14 days, treated wounds were fully closed. Blocking PDGFR-beta signaling did not prevent the differentiation of myofibroblasts in vitro but potently inhibited fibroblast proliferation and migration. In addition, PDGFR-beta inhibition in vivo was accompanied by abnormal microvascular morphogenesis reminiscent of that observed in PDGFR-beta-/- mice with significantly reduced immunostaining of the pericyte marker NG2. Imatinib treatment also inhibited pericyte proliferation and migration in vitro. This study highlights the significance of PDGFR-beta signaling for the recruitment, proliferation, and functional activities of fibro-blasts and pericytes during the early phases of wound healing.

Objective: To investigate expression of the chemokine receptor CCR2 on key cell types involved in the pathogenesis of systemic sclerosis (SSc) and to assess the potential for autocrine activation of SSc dermal fibroblasts via CCL2/CCR2., Methods: Chemokine receptor expression in skin biopsy tissues and explanted dermal fibroblasts from a well-characterized cohort of SSc patients was examined using immunohistochemistry and flow cytometry techniques. Autocrine regulation of the expression of fibrotic markers in CCR2+ SSc fibroblast cell lines was assessed using specific ligand or receptor antagonists., Results: We identified strong CCR2 expression in skin biopsy samples of early-stage diffuse cutaneous SSc (dcSSc), but not late-stage dcSSc or limited cutaneous SSc. Double labeling confirmed up-regulation of CCL2/CCR2 on myofibroblasts, pericytes, lymphocytes, macrophages, and endothelial cells. Explanted dermal fibroblasts from early dcSSc tissues expressed CCR2 and CXCR2 in 55% and 66% of cell strains, respectively. There was no expression in control fibroblasts. CCR2+ fibroblasts demonstrated a profibrotic phenotype, with overexpression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), and CCL2. Flow cytometric analysis identified a subset of CCR2+ SSc fibroblasts expressing the myofibroblast marker alpha-SMA. In these cultures, specific inhibition of CCL2 or CCR2 attenuated the overexpression of alpha-SMA, but not CTGF or plasminogen activator inhibitor 1., Conclusion: Our results show that CCR2 is up-regulated in early dcSSc on cell types known to be activated in the disease, which is consistent with a key role in SSc pathogenesis. CCR2 expression on SSc fibroblasts appears to regulate the expression of CCL2 and alpha-SMA. Our findings suggest potential autocrine regulation of key profibrotic properties via a CCL2/CCR2 loop in early-stage dcSSc.

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-beta (TGFbeta) receptor selectively on fibroblasts (TbetaRIIDeltak-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGFbeta signaling pathways in this strain and examine the profibrotic properties of explanted transgenic fibroblasts including myofibroblast differentiation and abnormal metalloproteinase production. Gene expression profiles of littermate wild type or transgenic fibroblasts were compared using high-density gene arrays and validated by Taqman reverse transcriptase-PCR, Northern and Western blotting. Using a specific inhibitor (SD-208) we demonstrate that the abnormal phenotype of these cells is dependent upon TbetaRI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGFbeta together with increased levels of wild type TbetaRII. Moreover, we confirm that transgene expression is itself regulated by TGFbeta and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGFbeta responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGFbeta1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of TbetaRIIDeltak-fib fibroblasts to exogenous TGFbeta1, despite their activated phenotype. Thus, transgenic fibroblasts recapitulate many hallmark biochemical properties of fibrotic cells, including high level CTGF (CCN2) expression and type I collagen overproduction, altered MMP production, and myofibroblast differentiation. These cells also show an enhanced ability to contract collagen gel matrices. Our study demonstrates that altered high affinity TGFbeta receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGFbeta overactivity in fibrosis.

Objective: To determine the gene-expression profile in dermal fibroblasts from type 1 tight-skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP-3) in Tsk1 mouse and human systemic sclerosis (SSc) skin., Methods: Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non-Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP-3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP-3 was explored in transgenic mouse fibroblasts and by transient transfection assays., Results: MCP-3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early-stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP-3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proalpha2(I) collagen promoter-reporter gene constructs were activated by MCP-3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP-3 could be diminished by a neutralizing antibody to transforming growth factor beta., Conclusion: We demonstrate, for the first time, overexpression of MCP-3 in early-stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.

The SMAD-mediated induction of connective tissue growth factor (CTGF), a fibroproliferative cytokine, by transforming growth factor (TGF)beta is required for the development of sustained fibrosis in humans. Here, we show that in fibroblasts, activation of the Ras/MEK/ERK pathway is required for the SMAD-mediated induction of CTGF by TGFbeta2. We then show that activation of protein kinase A (PKA) in fibroblasts is able to block Ras/MEK/ERK signaling and abolish the fibrotic response. Previously, we found that prostacyclin agonists were able to prevent the induction of CTGF in fibroblasts, and in patients with the fibrotic disease scleroderma. Here, we confirm the in vitro and in vivo antifibrotic effects of prostacyclin derivatives and show that these effects are due to PKA-dependent inhibition of the Ras/MEK/ERK pathway. Ras/MEK/ERK does not directly affect SMAD signaling. The coordinate and varied biological responses to TGFbeta are in part due to the interactions of signaling pathways within target cells. Specific inhibition of fibroblast Ras/MEK/ERK signaling might prevent fibrosis while leaving other physiological effects of TGFbeta unaltered.

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.

Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud's phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-beta to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-beta. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-beta.

Although the role of transforming growth factor beta (TGFbeta) in initiating fibrosis is well established, the role that TGFbeta plays in maintaining fibrosis is unclear. The gene encoding connective tissue growth factor (ccn2; ctgf), which promotes fibrosis, is not normally expressed in dermal fibroblasts unless TGFbeta is present. However, in dermal fibroblasts cultured from lesional areas of scleroderma, ccn2 (ctgf) is expressed constitutively. The contribution of several elements in the ccn2 (ctgf) promoter to basal and TGFbeta induced ccn2 (ctgf) expression in normal and scleroderma fibroblasts has been investigated. A functional SMAD binding site in the ccn2 (ctgf) promoter that is necessary for the TGFbeta mediated induction of this gene has been identified. The previously termed TGFbeta responsive enhancer (TGFbetaRE) in the ccn2 (ctgf) promoter has been found to be necessary for basal promoter activity in normal fibroblasts. The SMAD element is not necessary for the high ccn2 (ctgf) promoter activity seen in scleroderma fibroblasts. However, mutation of the previously termed TGFbetaRE reduces ccn2 (ctgf) promoter activity in scleroderma fibroblasts to that seen in normal fibroblasts. Thus, the maintenance of the scleroderma phenotype, as assessed by a high degree of ccn2 (ctgf) promoter activity, appears to be relatively independent of SMAD action and seems to reflect increased basal promoter activity.

In normal adult fibroblasts, transforming growth factor-beta (TGFbeta) induces the expression of connective tissue growth factor (CTGF). CTGF independently promotes fibroblast proliferation and matrix deposition, and in acute models of fibrosis promotes cell proliferation and collagen deposition acting synergistically with TGFbeta. In contrast to normal fibroblasts, fibroblasts cultured from fibrotic tissues express high basal levels of CTGF, even in the absence of added TGFbeta. Induction of transcription by TGFbeta requires the action of SMAD proteins. In this report we have investigated the role of SMADs in the TGFbeta-induction of CTGF in normal fibroblasts and in the elevated levels of CTGF expression found in dermal fibroblasts cultured from lesional areas of patients with scleroderma, a progressive fibrotic disorder that can affect all organs of the body. We have identified a functional SMAD binding site in the CTGF promoter. TGFbeta-induction of CTGF is dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent. However, mutation of the SMAD binding site does not reduce the high level of CTGF promoter activity observed in dermal fibroblasts cultured from lesional areas of scleroderma patients. Conversely, the previously termed TGFbetaRE in the CTGF promoter is required for basal CTGF promoter activity in normal fibroblasts and for the elevated level of CTGF promoter activity in scleroderma fibroblasts. Thus, the maintenance of the fibrotic phenotype in scleroderma fibroblasts, as visualized by excess CTGF expression, appears to be independent of SMAD-dependent TGFbeta signaling. Furthermore, given CTGF's activities, the high level of CTGF expression observed in scleroderma lesions may contribute to the excessive scarring observed in this disorder.

Objective: Reporter transgenes were introduced into the type 1 tight-skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast-specific genetic programs are activated in fibrosis., Methods: Transgenes harboring upstream fragments of the 5' flanking region of the mouse proalpha2(I) collagen gene (Col1a2), linked to a 400-bp minimal Col1a2 promoter driving an Escherichia coli beta-galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding. Expression of these transgenes, which function as lineage-specific markers of fibroblast differentiation, was compared between the Tsk-LacZ mice and non-Tsk littermates. Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor beta1 (TGFbeta1), was investigated by transient transfection of reporter constructs in tissue-culture cells., Results: There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age. This was maximal at 6 weeks old (mean +/- SD 237 +/- 24% of non-Tsk controls; P= 0.001). Recombinant TGFbeta1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk-LacZ fibroblasts showed elevated LacZ expression in tissue culture., Conclusion: These data suggest that activating signals in Tsk1/+ mice may act via fibroblast-specific regulatory elements within the murine Col1a2 gene. Although TGFbeta has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFbeta responsive in vitro, our results suggest that fibroblast-specific pathways may also be involved.

Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders, presumably secondary to the activation and production of transforming growth factor-beta (TGF-beta), a key inducer of fibroblast proliferation and matrix synthesis. The CTGF gene promoter has a TGF-beta response element that regulates its expression in fibroblasts but not epithelial cells or lymphocytes. Recent studies have shown that the macrophage-produced cytokine tumor necrosis factor alpha (TNFalpha) is necessary to promote inflammation and to induce genes, such as matrix metalloproteinases, involved with the early stages of wound healing. In this study, we examined the ability of TNFalpha to modulate CTGF gene expression. TNFalpha was found to suppress the TGF-beta-induced expression of CTGF protein in cultured normal fibroblasts. The activity of TNFalpha was blocked by NF-kappaB inhibitors. We showed that sequences between -244 and -166 of the CTGF promoter were necessary for both TGF-beta and TNFalpha to modulate CTGF expression. There was a constitutive expression of CTGF by scleroderma fibroblasts that was increased by TGF-beta treatment. Although TNFalpha was able to repress TGF-beta-induced CTGF and collagen synthesis both in normal and scleroderma skin fibroblasts, fibroblasts cultured from scleroderma patients were more resistant to TNFalpha as TNFalpha was unable to suppress the basal level of CTGF expression in scleroderma fibroblasts. Thus, we suspect that the high level of constitutive CTGF expression in scleroderma fibroblasts and its inability to respond to negative regulatory cytokines may contribute to the excessive scarring of skin and internal organs in patients with scleroderma.

Microvascular damage occurs in systemic sclerosis (SSc) and is associated with increased expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Elevated levels of the soluble circulating forms of these molecules have recently been reported in SSc. We have extended this observation by collecting serial serum samples from 12 patients with systemic sclerosis, at intervals between 4 and 12 months, through the course of their disease (mean period of observation 44 months). Circulating ICAM-1, VCAM-1 and E-selectin were measured by ELISA, and changes in these levels were compared with alterations in disease activity as assessed by skin sclerosis score, serum creatinine, erythrocyte sedimentation rate and pulmonary function tests coincident with each serum sample. The mean levels were ICAM-1 627 ng/ml, VCAM-1 959 ng/ml and E-selectin 81 ng/ml. In 8/12 patients, there was a substantial change in at least one disease parameter during the assessment period. In seven (88%) of these patients, changes in circulating VCAM-1 or E-selectin were associated with disease severity, falling with improvement in renal function or skin score, and rising with deterioration in pulmonary function tests. The maximum recorded level of VCAM-1 (3550 ng/ml) shortly preceded an acute renal SSc crisis. In two cases (25%), the correlation was statistically significant (P < or = 0.01). The ICAM-1 level did not reflect clinical changes in any patients. These results provide further evidence for endothelial cell dysfunction in SSc, and suggest that serial measurements of VCAM-1 and E-selectin may have potential value as surrogate markers for clinical progression or remission in this disease.