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Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1‐dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α‐smooth muscle actin (α‐SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF‐β under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR‐81, a G‐protein‐coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate‐mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR‐81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen‐deficient microenvironments.

In patients with pulmonary diseases such as idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome, progressive pulmonary fibrosis is caused by dysregulated wound healing via activation of fibroblasts after lung inflammation or severe damage. Migration of fibroblasts toward the fibrotic lesions plays an important role in pulmonary fibrosis. Fibrotic tissue in the lung is much stiffer than normal lung tissue. Emerging evidence supports the hypothesis that the stiffness of the matrix is not only a consequence of fibrosis, but also can induce fibroblast activation. Nevertheless, the effects of substrate rigidity on migration of lung fibroblasts have not been fully elucidated. We evaluated the effects of substrate stiffness on the morphology, α‐smooth muscle actin (α‐SMA) expression, and cell migration of primary human lung fibroblasts by using polyacrylamide hydrogels with stiffnesses ranging from 1 to 50 kPa. Cell motility was assessed by platelet‐derived growth factor (PDGF)‐induced chemotaxis and random walk migration assays. As the stiffness of substrates increased, fibroblasts became spindle‐shaped and spread. Expression of α‐SMA proteins was higher on the stiffer substrates (25 kPa gel and plastic dishes) than on the soft 2 kPa gel. Both PDGF‐induced chemotaxis and random walk migration of fibroblasts precultured on stiff substrates (25 kPa gel and plastic dishes) were significantly higher than those of cells precultured on 2 kPa gel. Transfection of the fibroblasts with short interfering RNA for α‐SMA inhibited cell migration. These findings suggest that fibroblast activation induced by a stiff matrix is involved in mechanisms of the pathophysiology of pulmonary fibrosis. It has been recognized that mechanical cues including matrix stiffness are involved in mechanisms underlying the pathogenesis of fibrosis. Here we report that as the stiffness of substrates increased, expression of α‐smooth muscle actin (SMA) and migrating properties of normal human lung fibroblasts increased. Moreover, transfection of fibroblasts with short interfering RNA for α‐SMA inhibited cell migration.

Hepatic fibrosis is a scarring process that is associated with an increased and altered deposition of extracellular matrix in liver. At the cellular and molecular level, this progressive process is mainly characterized by cellular activation of hepatic stellate cells and aberrant activity of transforming growth factor‐β1 and its downstream cellular mediators. Although the cellular responses to this cytokine are complex, the signalling pathways of this pivotal cytokine during the fibrogenic response and its connection to other signal cascades are now understood in some detail. Based on the current advances in understanding the pleiotropic reactions during fibrogenesis, various inhibitors of transforming growth factor‐β were developed and are now being investigated as potential drug candidates in experimental models of hepatic injury. Although it is too early to favour one of these antagonists for the treatment of hepatic fibrogenesis in human, the experimental results obtained yet provide stimulatory impulses for the development of an effective treatment of choice in the not too distant future. The present review summarises the actual knowledge on the pathogenesis of hepatic fibrogenesis, the role of transforming growth factor‐β and its signalling pathways in promoting the fibrogenic response, and the therapeutic modalities that are presently in the spotlight of many investigations and are already on the way to take the plunge into clinical studies.

The subpopulations of tumor pericytes undergo pathological phenotype switching, affecting their normal function in upholding structural stability and cross-communication with other cells. In the case of pancreatic ductal adenocarcinoma (PDAC), a significant portion of blood vessels are covered by an α-smooth muscle actin (αSMA)-expressing pericyte, which is normally absent from capillary pericytes. The DesminlowαSMAhigh phenotype was significantly correlated with intratumoral hypoxia and vascular leakiness. Using an in vitro co-culture system, we demonstrated that cancer cell-derived exosomes could induce ectopic αSMA expression in pericytes. Exosome-treated αSMA+ pericytes presented altered pericyte markers and an acquired immune-modulatory feature. αSMA+ pericytes were also linked to morphological and biomechanical changes in the pericyte. The PDAC exosome was sufficient to induce αSMA expression by normal pericytes of the healthy pancreas in vivo, and the vessels with αSMA+ pericytes were leaky. This study demonstrated that tumor pericyte heterogeneity could be dictated by cancer cells, and a subpopulation of these pericytes confers a pathological feature.

In passages above ten and growing very actively, we observed that some human lung fibroblasts cultured under standard conditions were transformed into a lineage of epithelial-like cells (ELC). To systematically evaluate the possible mesenchymal–epithelial transition (MET) occurrence, fibroblasts were obtained from normal lungs and also from lungs affected by idiopathic interstitial diseases. When an unusual epithelial-like phenotypic change was observed, cultured cells were characterized by confocal immunofluorescence microscopy, immunoblotting, immunocytochemistry, cytofluorometry, gelatin zymography, RT-qPCR, and hybridization in a whole-transcript human microarray. Additionally, microvesicles fraction (MVs) from ELC and fibroblasts were used to induce MET, while the microRNAs (miRNAs) contained in the MVs were identified. Pattern-gene expression of the original fibroblasts and the derived ELC revealed profound changes, upregulating characteristic epithelial-cell genes and downregulating mesenchymal genes, with a marked increase of E-cadherin, cytokeratin, and ZO-1, and the loss of expression of α-SMA, collagen type I, and Thy-1 cell surface antigen (CD90). Fibroblasts, exposed to culture media or MVs from the ELC, acquired ELC phenotype. The miRNAs in MVs shown six expressed exclusively in fibroblasts, and three only in ELC; moreover, twelve miRNAs were differentially expressed between fibroblasts and ELC, all of them but one was overexpressed in fibroblasts. These findings suggest that the MET-like process can occur in human lung fibroblasts, either from normal or diseased lungs. However, the biological implication is unclear.

Receptor for Advanced Glycation End Products Is Involved in Impaired Angiogenic Response in Diabetes Takuhito Shoji 1 , Hidenori Koyama 1 , Tomoaki Morioka 1 , Shinji Tanaka 1 , Akane Kizu 1 , Kohka Motoyama 1 , Katsuhito Mori 1 , Shinya Fukumoto 1 , Atsushi Shioi 2 , Noriko Shimogaito 3 , Masayoshi Takeuchi 3 , Yasuhiko Yamamoto 4 , Hideto Yonekura 4 , Hiroshi Yamamoto 4 and Yoshiki Nishizawa 1 1 Department of Metabolism, Endocrinology and Molecular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan 2 Department of Cardiovascular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan 3 Department of Pathophysiological Science, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan 4 Department of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan Address correspondence and reprint requests to Hidenori Koyama, MD, PhD, Department of Metabolism, Endocrinology and Molecular Medicine, (Second Department of Internal Medicine), Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail: hidekoyama{at}med.osaka-cu.ac.jp Abstract Angiogenic response is impaired in diabetes. Here, we examined the involvement of receptor for advanced glycation end products (RAGE) in diabetes-related impairment of angiogenesis in vivo. Angiogenesis was determined in reconstituted basement membrane protein (matrigel) plugs containing vascular endothelial growth factor (VEGF) implanted into nondiabetic or insulin-deficient diabetic wild-type or RAGE −/− mice. The total, endothelial, and smooth muscle (or pericytes) cells in the matrigel were significantly decreased in diabetes, with the regulation dependent on RAGE. In the matrigel, proangiogenic VEGF expression was decreased, while antiangiogenic thrombospondin-1 was upregulated in diabetic mice, regardless of the presence of RAGE. In wild-type mice, proliferating cell nuclear antigen (PCNA)-positive cells in the matrigel were significantly less in diabetic than in nondiabetic mice, while the numbers of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly higher. This alteration in PCNA- and TUNEL-positive cells in diabetes was not observed in RAGE −/− mice. Similarly, the percentage of nuclear factor κB–activated cells is enhanced in diabetes, with the regulation dependent on the presence of RAGE. Importantly, adenovirus-mediated overexpression of endogenous secretory RAGE, a decoy receptor for RAGE, restores diabetes-associated impairment of angiogenic response in vivo. Thus, RAGE appears to be involved in impairment of angiogenesis in diabetes, and blockade of RAGE might be a potential therapeutic target. αSM actin, α smooth muscle actin Ad-esRAGE, pAdHM15-esRAGE AGE, advanced glycation end product CML, N-carboxymethyllysine esRAGE, endogenous secretory RAGE MMP, matrix metalloproteinase NF-κB, nuclear factor-κB PCNA, proliferating cell nuclear antigen RAGE, receptor for AGE STZ, streptozotocin TSP, thrombospondin TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling VEGF, vascular endothelial growth factor Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted May 17, 2006. Received October 23, 2005. DIABETES

Expression of α-smooth muscle actin (αSMA) is constitutive in vascular smooth muscle cells, but is induced in nonmuscle cells such as hepatic stellate cells (HSCs). HSCs play important roles in both physiological homeostasis and pathological response. HSC activation is characterized by αSMA expression, which is regulated by the TGFβ-induced Smad pathway. Recently, protein kinase C (PKC) was identified to regulate αSMA expression. Diacylglycerol kinase (DGK) metabolizes a second-messenger DG, thereby controlling components of DG-mediated signaling, such as PKC. In the present study we aimed to investigate the putative role of DGKα in αSMA expression. Use of a cellular model indicated that the DGK inhibitor R59949 promotes αSMA expression and PKCδ phosphorylation. It also facilitates Smad2 phosphorylation after 30 min of TGFβ stimulation. Furthermore, immunocytochemical analysis revealed that DGK inhibitor pretreatment without TGFβ stimulation engenders αSMA expression in a granular pattern, whereas DGK inhibitor pretreatment plus TGFβ stimulation significantly induces αSMA incorporation in stress fibers. Through animal model experiments, we observed that DGKα-knockout mice exhibit increased expression of αSMA in the liver after 48 h of carbon tetrachloride injection, together with enhanced phosphorylation levels of Smad2 and PKCδ. Together, these findings suggest that DGKα negatively regulates αSMA expression by acting on the Smad and PKCδ signaling pathways, which differentially regulate stress fiber incorporation and protein expression of αSMA, respectively.

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Aims There are few studies on the treatment of heart failure by injecting stem cells into the pericardial cavity. Can the cells injected into the pericardial cavity migrate through the epicardium to the myocardial tissue? Whether there is therapeutic effect and the mechanism of therapeutic effect are still unclear. This study investigated the therapeutic efficacy and evidence of cell migration of adipose‐derived stem cells (ADSCs) injected into the pericardial cavity in rat heart failure. The aim of this study is to demonstrate the effectiveness and mechanism of treating heart failure by injecting stem cells into the pericardial cavity, laying an experimental foundation for a new approach to stem cell therapy for heart disease in clinical practice. Methods and results The inguinal adipose tissue of male SD rats aged 4–6 weeks was taken, ADSCs were isolated and cultured, and their stem cell surface markers were identified. Forty rats aged 6–8 weeks were divided into sham operation group, heart failure group, and treatment group; there were 15 rats in the heart failure group and 15 rats in the treatment group. The heart failure model was established by intraperitoneal injection of adriamycin hydrochloride. The heart function of the three groups was detected by small animal ultrasound. The model was successful if the left ventricular ejection fraction < 50%. The identified ADSCs were injected into the pericardial cavity of rats in the treatment group. The retention of transplanted cells in pericardial cavity was detected by small animal in vivo imaging instrument, and the migration of transplanted cells into myocardial tissue was observed by tissue section and immunofluorescence. Western blotting and immunohistochemical staining were used to detect brain natriuretic peptide (BNP), α‐smooth muscle actin (α‐SMA), and C‐reactive protein (CRP). ADSCs express CD29, CD44, and CD73. On the fourth day after injection of ADSCs into pericardial cavity, they migrated to myocardial tissue through epicardium and gradually diffused to deep myocardium. The cell density in the pericardial cavity remains at a high level for 10 days after injection and gradually decreases after 10 days. Compared with the heart failure group, the expression of BNP and α‐SMA decreased (P < 0.05 and P < 0.001, respectively), and the expression of CRP in the treatment group was higher than that in the heart failure group (P < 0.0001). A small amount of BNP, α‐SMA, and CRP was expressed in the myocardium of the sham operation group. After injection of ADSCs, interleukin‐6 in myocardial tissue was significantly lower than that in heart failure myocardium (P < 0.01). After treatment, vascular endothelial growth factor A was significantly higher than that of heart failure (P < 0.01). Conclusions Pericardial cavity injected ADSCs can penetrate the epicardium, migrate into the myocardium, and have a therapeutic effect on heart failure. Their mechanism of action is to exert therapeutic effects through anti‐inflammatory, anti‐fibrosis, and increased angiogenesis.

The processes involved in regeneration of cutaneous compared to corneal tissues involve different intrinsic mechanisms. Importantly, cutaneous wounds involve healing by angiogenesis but vascularization of the cornea obscures vision. Previous studies showed that topically applied calreticulin (CALR) healed full‐thickness excisional animal wounds by a tissue regenerative process markedly enhancing repair without evoking angiogenesis. In the current study, the application of CALR in a rabbit corneal injury model: (1) accelerated full wound closure by 3 days (2) accelerated delayed healing caused by corticosteroids, routinely used to prevent post‐injury inflammation, by 6 days and (3) healed wounds without vascularization or fibrosis/hazing. In vitro, CALR stimulated proliferation of human corneal epithelial cells (CE) and corneal stromal cells (keratocytes) by 1.5‐fold and 1.4‐fold, respectively and induced migration of CE cells and keratocytes, by 72% and 85% compared to controls of 44% and 59%, respectively. As a marker of decreased fibrosis, CALR treated corneal wounds showed decreased immunostaining for α‐smooth muscle actin (α‐SMA) by keratocytes and following CALR treatment in vitro, decreased the levels of TGF‐β2 in human CE cells and α‐SMA in keratocytes. CALR has the potential to be a novel therapeutic both, to accelerate corneal healing from various injuries and in conjunction with corticosteroids.