Connective Tissue Growth Factor Secretion Research Articles

Activin A Is a Master Regulator of Phenotypic Switch in Adipose Stromal Cells Initiated by Activated Immune Cell-Secreted Interleukin-1β.

Prolonged tissue ischemia and inflammation lead to organ deterioration and are often accompanied by microvasculature rarefaction, fibrosis, and elevated systemic Activin A (ActA), the level of which frequently correlates with disease severity. Mesenchymal stromal cells are prevalent in the perivascular niche and are likely involved in tissue homeostasis and pathology. This study investigated the effects of inflammatory cells on modulation of phenotype of adipose mesenchymal stromal cells (ASC) and the role of ActA in this process. Peripheral blood mononuclear cells were activated with lipopolysaccharide (activated peripheral blood mononuclear cells [aPBMC]) and presented to ASC. Expression of smooth muscle/myofibroblast markers, ActA, transforming growth factors beta 1-3 (TGFβ1-3), and connective tissue growth factor (CTGF) was assessed in ASC. Silencing approaches were used to dissect the signaling cascade of aPBMC-induced acquisition of myofibroblast phenotype by ASC. ASC cocultured with aPBMC or exposed to the secretome of aPBMC upregulated smooth muscle cell markers alpha smooth muscle actin (αSMA), SM22α, and Calponin I; increased contractility; and initiated expression of ActA. Interleukin (IL)-1β was sufficient to replicate this response, whereas blocking IL-1β eliminated aPBMC effects. ASC-derived ActA stimulated CTGF and αSMA expression in ASC; the latter independent of CTGF. Induction of αSMA in ASC by IL-1β or ActA-enriched media relied on extracellular enzymatic activity. ActA upregulated mRNA levels of several extracellular matrix proteins in ASC, albeit to a lesser degree than TGFβ1, and marginally increased cell contractility. In conclusion, the study suggests that aPBMC induce myofibroblast phenotype with weak fibrotic activity in perivascular progenitors, such as ASC, through the IL-1β-ActA signaling axis, which also promotes CTGF secretion, and these effects require ActA extracellular enzymatic processing.

Extracellular Matrix Stiffness Regulates Microvascular Stability by Controlling Endothelial Paracrine Signaling to Determine Pericyte Fate.

The differentiation of pericytes into myofibroblasts causes microvascular degeneration, ECM (extracellular matrix) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel 2-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Primary microvessels were cultured in the TGM2D medium (tubular microvascular growth medium on 2-dimensional substrates). Stiff ECM was prepared by incubating ECM solution in regular culture dishes for 1 hour followed by PBS wash. Soft ECM with Young modulus of ≈6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, RT-qPCR (real-time quantitative polymerase chain reaction), Western blotting, and knockdown experiments were performed on the cells. Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ (transforming growth factor beta) signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined 2 pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As integrins are the major mechanosensor, we performed RT-qPCR screening of integrin family members and found Itgb1 (integrin β1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF (connective tissue growth factor), to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. ECM stiffness and TGFβ signaling cooperatively regulate microvascular stability and pericyte-myofibroblast differentiation. Stiff ECM promotes EC ITGB1 phosphorylation (Y783) and CTGF secretion, which induces pericyte-myofibroblast differentiation.

Effect of Baricitinib on the epithelial-mesenchymal transition of alveolar epithelial cells induced by IL-6

ObjectiveInterstitial lung disease (ILD) is one of the common complications of Connective tissue disease (CTD). Epithelial-mesenchymal transition (EMT) is one of the main pathological mechanisms of ILD. IL-6 may induce ILD through the JAK/STAT pathway. Therefore, exploring the mechanism of IL-6 on the EMT of alveolar epithelial cells and inhibition JAK/STAT pathway with Baricitinib on the EMT of alveolar epithelial cells is helpful in revealing the pathogenesis of CTD-ILD and guiding treatment. MethodsElectrochemiluminescence was applied to detect the changes in serum IL-6 levels before and after treatment in 37 patients with anti-synthetase syndrome-associated ILD; A549 cells (a human AEC cell line) were incubated with IL-6, Baricitinib, or both IL-6 and Baricitinib, and changes in EMT-related markers levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT signaling pathways were examined by western blot. The level of Connective tissue growth factor (CTGF) and Hydroxyproline (Hyp) in cell supernatants was measured by ELISA. ResultsSerum IL-6 level in patients with anti-synthetase syndrome-associated ILD was significantly higher than that in health (6.78(4.19, 16.14)pg/ml vs. 2.10(1.43, 5.18)pg/ml, p < 0.01). The level of IL-6 in the improvement group of ASS-ILD was considerably decreased than that before treatment(before(7.48(4.54, 22.76) pg/mL vs. 5.00(3.46, 11.32)pg/mL, p < 0.01), p < 0.01), and the level of IL-6 in the progressive group of ASS-ILD was significantly higher than that before treatment(before(7.49(6.77, 35.80) pg/mL vs. 30.02(8.01, 82.98) pg/mL, p < 0.05). IL-6 increased the expression of epithelial phenotypic marker E-cadherin and inhibited mesenchymal phenotypic markers, including vimentin and N-cadherin in A549 cells. Moreover, IL-6-induced EMT was attenuated by Baricitinib. Furthermore, we found that IL-6 activated the phosphorylation of JAK1/2, STAT3, and Baricitinib, partially inhibiting these changes in this process. Baricitinib reduced the secretion of CTGF and Hyp in A549 cells. ConclusionThe significant higher level of IL-6 in patients with anti-synthase syndrome-associated ILD may be related to disease activity and recurrence. Our results suggest that Baricitinib attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the presence of IL-6 through the JAK/STAT signaling pathway.

Bone marrow mesenchymal stem cell sheets with high expression of hBD3 and CTGF promote periodontal regeneration

The multi-bacterial environment of the oral cavity makes it hard for periodontal regeneration. As a class of antimicrobial peptide, beta defensin has been found to show broad-spectrum antibacterial ability. In addition, connective tissue growth factor (CTGF) is demonstrated to play a great role in multi-physiological events such as angiogenesis, wound healing and, more importantly, fibrogenesis. In this study, human β defensin 3 (hBD3) and CTGF were co-transfected into bone marrow derived mesenchymal stem cells (BMSCs) for preparing cell sheets. The transfection efficiency was detected through fluorescence of eGFP and western blot assay. Our results showed that the hBD3 and CTGF proteins were highly and stably expressed in the BMSCs after transfection. The results of RT-PCR and induced differentiation indicated that hBD3 promoted osteogenic differentiation of BMSCs, while CTGF significantly increased fibrogenic differentiation even in the presence of hBD3. The BMSCs acquired stronger capacity in terms of promoting M2 polarization of RAW 264.7 macrophages fulfilled by the transfection and secretion of hBD3 and CTGF. To further evaluate the periodontal remodeling performance of cell sheets, a coralline hydroxyapatite (CHA)-chitosan based hydrogel-human tooth system was designed to simulate the natural periodontal environment. The results showed that dense extracellular matrix, oriented fiber arrangement, and abundant collagen deposition appeared in the area of BMSCs sheets after subcutaneous transplantation. Altogether, our data showed that the lentivirus transfected BMSCs sheets had a promising application prospect for periodontal repair.

Endothelial cell-derived pro-fibrotic factors increase TGF-β1 expression by smooth muscle cells in response to cycles of hypoxia-hyperoxia

BackgroundThe vascular pathology of peripheral artery disease (PAD) encompasses abnormal microvascular architecture and fibrosis in response to ischemia-reperfusion (I/R) cycles. We aimed to investigate the mechanisms by which pathological changes in the microvasculature direct fibrosis in the context of I/R. MethodsPrimary human aortic endothelial cells (ECs) were cultured under cycles of normoxia-hypoxia (NH) or normoxia-hypoxia-hyperoxia (NHH) to mimic I/R. Primary human aortic smooth muscle cells (SMCs) were cultured and treated with media from the ECs. FindingsThe mRNA and protein expression of the pro-fibrotic factors platelet derived growth factor (PDGF)-BB and connective tissue growth factor (CTGF) were significantly upregulated in ECs undergoing NH or NHH cycles. Treatment of SMCs with media from ECs undergoing NH or NHH cycles led to significant increases in TGF-β1, TGF-β pathway signaling intermediates, and collagen expression. Addition of neutralizing antibodies against PDGF-BB and CTGF to the media blunted the increases in TGF-β1 and collagen expression. Treatment of SMCs with PAD patient-derived serum also led to increased TGF-β1 levels. InterpretationIn an in-vitro model of I/R, which recapitulates the pathophysiology of PAD, increased secretion of PDGF-BB and CTGF by ECs was shown to be predominantly driving TGF-β1-mediated expression by SMCs. These cell culture experiments help elucidate the mechanism and interaction between ECs and SMCs in microvascular fibrosis associated with I/R. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of I/R. FundingNational Institute on Aging at the National Institutes of Health grant number R01AG064420. Research in contextEvidence before this study: Previous studies in gastrocnemius biopsies from peripheral artery disease (PAD) patients showed that transforming growth factor beta 1 (TGF-β1), the most potent inducer of pathological fibrosis, is increased in the vasculature of PAD patients and correlated with collagen deposition. However, the exact cellular source of TGF-β1 remained unclear. Added value of this study: Exposing cells to cycles of normoxia-hypoxia-hyperoxia (NHH) resulted in pathological changes that are consistent with human PAD. This supports the idea that the use of NHH may be a reliable, novel in vitro model of PAD useful for studying associated pathophysiological mechanisms. Furthermore, pro-fibrotic factors (PDGF-BB and CTGF) released from endothelial cells were shown to induce a fibrotic phenotype in smooth muscle cells. This suggests a potential interaction between these cell types in the microvasculature that drives increased TGF-β1 expression and collagen deposition. Thus, targeting these pro-fibrotic factors may be an effective strategy to combat fibrosis in response to cycles of ischemia-reperfusion.

Cyclic Stretch Induces Vascular Smooth Muscle Cells to Secrete Connective Tissue Growth Factor and Promote Endothelial Progenitor Cell Differentiation and Angiogenesis

Endothelial progenitor cells (EPCs) play a vital role in endothelial repair following vascular injury by maintaining the integrity of endothelium. As EPCs home to endothelial injury sites, they may communicate with exposed vascular smooth muscle cells (VSMCs), which are subjected to cyclic stretch generated by blood flow. In this study, the synergistic effect of cyclic stretch and communication with neighboring VSMCs on EPC function during vascular repair was investigated. In vivo study revealed that EPCs adhered to the injury site and were contacted to VSMCs in the Sprague–Dawley (SD) rat carotid artery injury model. In vitro, EPCs were cocultured with VSMCs, which were exposed to cyclic stretch at a magnitude of 5% (which mimics physiological stretch) and a constant frequency of 1.25 Hz for 12 h. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cells (ECs) and promoted angiogenesis. Meanwhile, cyclic stretch upregulated the mRNA expression and secretion level of connective tissue growth factor (CTGF) in VSMCs. Recombinant CTGF (r-CTGF) treatment promoted endothelial differentiation of EPCs and angiogenesis, and increased their protein levels of FZD8 and β-catenin. CTGF knockdown in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of the FZD8/β-catenin signaling pathway. FZD8 knockdown repressed endothelial differentiation of EPCs and their angiogenic activity. Wnt signaling inhibitor decreased the endothelial differentiation and angiogenetic ability of EPCs cocultured with stretched VSMCs. Consistently, an in vivo Matrigel plug assay demonstrated that r-CTGF-treated EPCs exhibited enhanced angiogenesis; similarly, stretched VSMCs also induced cocultured EPC differentiation toward ECs. In a rat vascular injury model, r-CTGF improved EPC reendothelialization capacity. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which, in turn, activates FZD8 and β-catenin to promote both differentiation of cocultured EPCs into the EC lineage and angiogenesis, suggesting that CTGF acts as a key intercellular mediator and a potential therapeutic target for vascular repair.

Lysophosphatidic acid as a regulator of endometrial connective tissue growth factor and prostaglandin secretion during estrous cycle and endometrosis in the mare

BackgroundEquine endometrosis is a chronic degenerative condition, described as endometrial fibrosis that forms in the stroma, under the basement membrane and around the endometrial glands. The role of lysophosphatidic acid (LPA) in the development of tissue fibrosis varies depending on the organ, and its profibrotic role in mare endometrosis remains unclear. The study aimed to establish the endometrial presence of LPA and its receptors (LPAR1–4), together with its effects on connective tissue growth factor (CTGF) and prostaglandins (PG) secretion from equine endometrium under physiological (estrous cycle), or pathological conditions (endometrosis). Mare endometria in the mid-luteal phase (n = 5 for each category I, IIA, IIB, III of Kenney and Doig) and in the follicular phase (n = 5 for each category I, IIA, III and n = 4 for IIB) were used. In experiment 1, the levels of LPA, LPAR1–4 mRNA level and protein abundance were investigated in endometria at different stages of endometrosis. In experiment 2, the in vitro effect of LPA (10− 9 M) on the secretion of CTGF and PGs from endometrial tissue explants at different stages of endometrosis were determined.ResultsEndometrial LPA concentration was higher in the mid-luteal phase compared to the follicular phase in category I endometrium (P < 0.01). There was an alteration in endometrial concentrations of LPA and LPAR1–4 protein abundance in the follicular phase at different stages of endometrosis (P < 0.05). Additionally, LPA increased the secretion of PGE2 from category I endometrium in both phases of the estrous cycle (P < 0.05). The effect of LPA on the secretion of CTGF and PGF2α from endometrial tissue was altered depending on different stages of endometrosis (P < 0.05).ConclusionOur data indicate that endometrosis disturbs proper endometrial function and is associated with altered endometrial LPA concentration, its receptor expression and protein abundance, PGE2/PGF2α ratio, and CTGF secretion in response to LPA. These changes could influence several physiological events occurring in endometrium in mare during estrous cycle and early pregnancy.

Cyclic stretch induces vascular smooth muscle cell-derived connective tissue growth factor secretion and promotes endothelial progenitor cell differentiation and angiogenesis

Endothelial progenitor cells (EPCs) play an important role in endothelial repair following vascular injury and maintaining the integrity of endothelium, but the underlying molecular mechanisms regulating the differentiation of EPCs remain to be studied. In this study, the effects of cyclic stretch and the neighboring vascular smooth muscle cells (VSMCs) on the differentiation of EPCs, as well as the underlying mechanism were investigated in a co-culture model. Bone marrow-derived EPCs were cocultured with VSMCs, which were exposed to cyclic stretch with a magnitude of 5% (which mimics physiological mechanical stress) at a constant frequency of 1.25 Hz. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cell (ECs), with upregulation of EC markers (CD31, vWF and KDR) and promoted angiogenesis. Meanwhile, cyclic stretch unregulated the mRNA level and secretion level of connective tissue growth factor (CTGF). After treated with recombinant CTGF (r-CTGF) at the concentration of 20 ng/ml, the mRNA expression of CD31, vWF and KDR in EPCs were increased, EPC tube formation and the protein level of FZD8 and β-catenin were promoted. Small interfering RNA (siRNA)-mediated knockdown of CTGF in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of FZD8/β-catenin signaling pathway in vitro. Consistently, in vivo matrigel plug assay demonstrated that r-CTGF treated EPCs enhanced angiogenesis. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which in turn activates FZD8 and β-catenin to promote co-cultured EPC differentiation into EC lineage and angiogenesis. Our findings provide insights into the mechanisms underlying the VSMC/EPC interactions and the pro-angiogenic function of cyclic stretch in modulating EPC differentiation. CTGF acts as a key intercellular mediator and may be as a potential therapeutic target for vascular injury.

Tspan15 Is a New Stemness-Related Marker in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second cause of cancer-related deaths worldwide. A clearer understanding of the molecular mechanisms underlying tumor growth and invasiveness remains crucial for developing new therapies. Here, the expression of tetraspanins, a family of plasma membrane organizers involved in tumor progression, has been addressed. Integrative approaches combining transcriptomics and bioinformatics allow demonstrating the induced and heterogeneous expression of Tspan15 in HCC. Tspan15 positive tumors exhibit signatures related to hepatic progenitor cells as well as recurrence of cancer. Immunohistochemistry experiments confirm Tspan15 expression in the subset of HCC expressing stemness-related markers such as EpCAM and Cytokeratin-19. Functional networks reveal that most of these genes expressed in correlation to Tspan15 support cell proliferation. Furthermore, Tspan15 overexpression in the hepatoma cell line HepG2 significantly increases cell proliferation. A quantitative proteomic analysis of the secretome reveals a higher abundance of the protein connective tissue growth factor (CTGF), a pleiotropic matricellular signaling protein. Proteomic profiling of Tspan15 complexes allows identifying numerous membrane proteins including several growth factor receptors. Finally, Tspan15 increases ERK1/2 phosphorylation that directly controls CTGF expression and secretion. In conclusion, Tspan15 is a new stemness-related marker in HCC which exhibits high potential of tumor growth and recurrence.

TGFβ mediates collagen production in human CRSsNP nasal mucosa-derived fibroblasts through Smad2/3-dependent pathway and CTGF induction and secretion.

Chronic rhinosinusitis without nasal polyp (CRSsNP) is characterized by tissue remodeling and fibrosis. Transforming growth factor-β (TGF-β) is considered a master switch in the induction of the profibrotic program which can induce fibroblasts to synthesize and contract extracellular matrix (ECM) proteins. A previous study has shown TGF-β1 signaling and collagen overproduction in the CRSsNP, but the responsible cells and mechanism of action remain unclear. Therefore, this study was aimed to investigate the relationship between TGF-β1 stimulation and collagen expression and to explore the role of connective tissue growth factor (CTGF) during the remodeling process using human CRSsNP nasal mucosa tissues and mucosa-derived fibroblasts as main materials. We found that TGF-β1 and its isoforms could promote collagen protein expression. Concomitantly, TGF-β1 caused CTGF expression and secretion. An addition of exogenous CTGF to fibroblasts also caused collagen expression. In accordance with these observations, TGF-β1, CTGF, and collagen were highly expressed in the subepithelial stroma region of CRSsNP nasal mucosa, as determined by immunohistochemistry. The TGF-β1-mediated collagen expression could be blocked by actinomycin D and SIS3, suggesting that the induction was through transcriptional regulation and Smad2/3-dependent pathway. Finally, we demonstrated that CTGF small interfering RNA knockdown led to a substantial decrease in TGF-β1-mediated collagen expression. Collectively, our results provide first and further evidence that TGF-β1 mediates collagen expression-production through a canonical Smad2/3-dependent pathway and CTGF induction and secretion in human nasal fibroblasts. Moreover, TGF-β1, CTGF, and collagen are highly expressed in human CRSsNP nasal mucosa specimens, suggesting their roles in tissue remodeling during CRSsNP progression.

Deregulation of Hippo-TAZ pathway during renal injury confers a fibrotic maladaptive phenotype.

Although yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), nuclear transducers of the Hippo pathway, are mostly silent in adult organs, aberrant activation of YAP/TAZ promotes tumorigenesis and abnormal tissue repair. The extent of involvement of TAZ in chronic kidney disease (CKD) is unknown. In our study, increased TAZ nuclear accumulation and expression in the tubulointerstitium was readily evident in 3 models of renal injury including obstructive, aristolochic acid (AA), and diabetic nephropathy, correlating with fibrosis progression. Stable TAZ overexpression in human kidney (HK)-2 epithelial cells promoted connective tissue growth factor (CTGF), fibronectin, vimentin, and p21 expression, epithelial dedifferentiation, and growth inhibition, in part, via Sma mothers against decapentaplegic homologue (SMAD)-3-dependent CTGF induction. CTGF secretion by TAZ-overexpressing epithelium also triggered proliferative defects in nonengineered HK-2 cells confirming a nonautonomous role of TAZ ( via a paracrine mechanism) in orchestrating kidney epithelial cell-cell communication. Renal tubular-specific induction of TGF-β1 in mice and TGF-β1 stimulation of HK-2 cells resulted in TAZ protein up-regulation. TAZ stable silencing in HK-2 cells abrogated TGF-β1-induced expression of target genes without affecting SMAD3 phosphorylation, which is also crucial for fibrotic reprogramming. Thus, TAZ was activated in fibrosis through TGF-β1-dependent mechanisms and sustained TAZ signaling promotes epithelial maladaptive repair. TAZ is also a novel non-SMAD downstream effector of renal TGF-β1 signaling, establishing TAZ as a new antifibrosis target for treatment of CKD.-Anorga, S., Overstreet, J. M., Falke, L. L., Tang, J., Goldschmeding, R. G., Higgins, P. J., Samarakoon, R. Deregulation of Hippo-TAZ pathway during renal injury confers a fibrotic maladaptive phenotype.

Sustained β-AR stimulation induces synthesis and secretion of growth factors in cardiac myocytes that affect on cardiac fibroblast activation

Paracrine factors, including growth factors and cytokines, released from cardiac myocytes following β-adrenergic receptor (β-AR) stimulation regulate cardiac fibroblasts. Activated cardiac fibroblasts have the ability to increase collagen synthesis, cell proliferation and myofibroblast differentiation, leading to cardiac fibrosis. However, it is unknown which β-AR subtypes and signaling pathways mediate the upregulation of paracrine factors in cardiac myocytes. In this study, we demonstrated that sustained stimulation of β-ARs significantly induced synthesis and secretion of growth factors, including connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), via the cAMP-dependent and protein kinase A (PKA)-dependent pathways. In addition, isoproterenol (ISO)-mediated synthesis and secretion of CTGF and VEGF through the β1-AR and β2-AR subtypes. Paracrine factors released by cardiac myocytes following sustained β-AR stimulation are necessary for the induction of cell proliferation and synthesis of collagen I, collagen III and α-smooth muscle actin (α-SMA) in cardiac fibroblasts, confirming that β-AR overstimulation of cardiac myocytes induces cardiac fibrosis by releasing several paracrine factors. These effects can be antagonized by β-blockers, including atenolol, metoprolol, and propranolol. Thus, the use of β-blockers may have beneficial effects on the treatment of myocardial fibrosis in patients with heart failure.

Sitagliptin ameliorates high glucose-induced cell proliferation and expression of the extracellular matrix in glomerular mesangial cells

Diabetic nephropathy (DN) is one of the most important causes that leads to end-stage renal disease and the efficacy of strategies currently available for the prevention of DN remains unsatisfactory. Sitagliptin (SIT), which is a dipeptidyl peptidase-4 inhibitor, exhibited a modest beneficial effect on glycated hemoglobin levels and is capable of ameliorating renal ischemia reperfusion injury. By determining the expression of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), collagen type IV (ColIV) and fibronectin (FN) levels in high glucose-cultured glomerular mesangial cells (MCs), the present study aimed to assess the anti-proliferative and anti-fibrotic effects of SIT on the therapeutic potential for the prevention of DN and its mechanism. Specifically, cell proliferation was determined via cell counting kit-8 assay, and the expression levels of TGF-β1 and CTGF mRNA were detected by reverse transcription polymerase chain reaction analysis. Furthermore, the secretion of TGF-β1, CTGF, ColIV and FN proteins was measured via enzyme-linked immunosorbent assays. The results demonstrated that high glucose induced the proliferation of MCs and enhanced the expression of TGF-β1, CTGF, ColIV and FN. Furthermore, treatment with SIT inhibited cell proliferation and the expression of TGF-β1, CTGF, ColIV and FN induced by high glucose. In conclusion, SIT inhibits cell proliferation and the expression of the major extracellular matrix proteins induced by high glucose, indicating its value for treating or relieving DN.

Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Age‐related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient‐specific, stem cell‐derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC‐derived CEC‐like cells. RNA‐seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)‐related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF‐driven iPSC‐derived CEC‐like cells formed capillary tube‐like vascular networks, and expressed the EC‐specific markers CD31, ICAM1, PLVAP, vWF, and the CEC‐restricted marker CA4. In combination with RPE and photoreceptor cells, patient‐specific iPSC derived CEC‐like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533–1546

Neuronal CTGF/CCN2 negatively regulates myelination in a mouse model of tuberous sclerosis complex.

Disruption of myelination during development has been implicated in a range of neurodevelopmental disorders including tuberous sclerosis complex (TSC). TSC patients with autism display impairments in white matter integrity. Similarly, mice lacking neuronal Tsc1 have a hypomyelination phenotype. However, the mechanisms that underlie these phenotypes remain unknown. In this study, we demonstrate that neuronal TSC1/2 orchestrates a program of oligodendrocyte maturation through the regulated secretion of connective tissue growth factor (CTGF). We characterize oligodendrocyte maturation both in vitro and in vivo. We find that neuron-specific Tsc1 deletion results in an increase in CTGF secretion that non-cell autonomously stunts oligodendrocyte development and decreases the total number of oligodendrocytes. Genetic deletion of CTGF from neurons, in turn, mitigates the TSC-dependent hypomyelination phenotype. These results show that the mechanistic target of rapamycin (mTOR) pathway in neurons regulates CTGF production and secretion, revealing a paracrine mechanism by which neuronal signaling regulates oligodendrocyte maturation and myelination in TSC. This study highlights the role of mTOR-dependent signaling between neuronal and nonneuronal cells in the regulation of myelin and identifies an additional therapeutic avenue for this disease.

P38 pathway as a key downstream signal of connective tissue growth factor to regulate metastatic potential in non‐small‐cell lung cancer

Although the secretory matricellular protein connective tissue growth factor (CTGF) has been reported to be related to lung cancer metastasis, the precise mechanism by which CTGF regulates lung cancer metastasis has not been elucidated. In the present study, we show the molecular link between CTGF secretion and the p38 pathway in the invasive and metastatic potential of non‐small‐cell lung cancer (NSCLC). Among three different human NSCLC cell lines (PC‐14, A549, and PC‐9), their in vitro invasiveness was inversely correlated with the level of CTGF secretion. By supplementing or reducing CTGF secretion in NSCLC culture, dysregulation of the invasive and metastatic potential of NSCLC cell lines was largely compensated. By focusing on the protein kinases that are known to be regulated by CTGF, we found that the p38 pathway is a key downstream signal of CTGF to regulate the metastatic potential of NSCLC. Importantly, a negative correlation between CTGF and phosphorylation status of p38 was identified in The Cancer Genome Atlas lung adenocarcinoma dataset. In the context of the clinical importance of our findings, we showed that p38 inhibitor, SB203580, reduced the metastatic potential of NSCLC secreting low levels of CTGF. Collectively, our present findings indicate that the CTGF/p38 axis is a novel therapeutic target of NSCLC metastasis, particularly NSCLC secreting low levels of CTGF.

The Properties of 3 Different Plasma Formulations and Their Effects on Tendinopathic Cells

Background: Tendinopathies are attributed to failure of the healing process and inadequate tissue remodeling. Plasma injections can trigger regenerative responses by modifying the molecular microenvironment. Purpose: To examine the differences in the mitotic, chemotactic, anabolic, and inflammatory effects between leukocyte- and platelet-rich plasma (L-PRP), platelet-rich plasma (PRP), and platelet-poor plasma (PPP). Study Design: Controlled laboratory study. Methods: Tendinopathic cells were cultured in 3-dimensional (3D) hydrogels formed using PPP, PRP, and L-PRP. Cell migration was evaluated using a μ-Slide chemotaxis chamber with video microscopy. Proliferation was assessed using XTT assays. Expression of genes associated with matrix turnover, including type 1 collagen (COL1A1), COL3A1, aggrecan, decorin, fibronectin, matrix metalloproteinase 1 (MMP-1), MMP-3, A Disintegrin-Like And Metalloprotease With Thrombospondin Type 1 Motif proteins 4 (ADAMTS-4), and ADAMTS-5, was assessed using real-time reverse-transcription polymerase chain reaction. Secreted inflammatory proteins, including interleukin (IL)–1β, IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES), as well as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF), were quantified using enzyme-linked immunosorbent assay. Results: Tendinopathic cells migrate at a higher velocity along L-PRP and PRP than along PPP gradients. PRP and L-PRP promote hypercellularity. PPP and PRP showed more pronounced anabolic properties, as demonstrated by enhanced COL1A1 and COL3A1 and reduced MMP-1 expression. Decorin, fibronectin, and aggrecan were downregulated in L-PRP compared with PPP and PRP. L-PRP and PRP were shown to be more proinflammatory than PPP in terms of IL-6 secretion, but cells in PPP showed MCP-1high phenotype. CTGF secretion was significantly reduced in L-PRP compared with PPP and PRP. Conclusion: The main advantages of L-PRP and PRP use, compared with PPP, include their stronger chemotactic and proliferative properties. While PPP and PRP stimulate matrix anabolism, L-PRP is more proinflammatory. Emphasis should be placed on the temporal needs and biological characteristics of injured tendons, and plasma formulations need to be tailored accordingly. Clinical Relevance: Versatile systems allowing the preparation of different plasma formulations, such as PPP, PRP, or L-PRP, can help refine clinical applications by taking advantage of their different biological properties.

Inhibitors of oxygen sensing prolyl hydroxylases regulate nuclear localization of the transcription factors Smad2 and YAP/TAZ involved in CTGF synthesis

Pharmacological inhibition of oxygen sensing prolyl hydroxylase domain enzymes (PHDs) has been shown to preserve renal structure and function in various models of kidney disease. Since transforming growth factor β-1 (TGFβ-1) is one of the major mediators of kidney injury, we investigated if inhibition of PHDs with subsequent stabilization of hypoxia inducible transcription factors (HIF) might interfere with TGFβ-1 signaling with special emphasis on its target gene connective tissue growth factor (CTGF).Overnight incubation of human renal tubular cells, primary cells and cell lines, with the PDH inhibitor DMOG increased Smad3 expression, but barely affected Smad2. Both Smads were translocated into the nucleus upon activation of the cells with TGFβ-1. Interestingly, Smad3 nuclear localization was enhanced upon pretreatment of the cells with DMOG for several hours, whereas nuclear Smad2 was reduced. This differential localization was independent of Smad2/3 phosphorylation. Reduced nuclear Smad2 correlated with impaired CTGF secretion in DMOG-treated cells and transient downregulation of Smad2 interfered with TGFβ-1-induced CTGF synthesis. Furthermore, YAP was confirmed as indispensable transcription factor involved in CTGF synthesis. Nuclear localization of YAP and TAZ was reduced in DMOG-treated cells.Our data thus provide evidence for DMOG-mediated reduction of CTGF expression by regulating the nuclear localization of the transcription factors Smad2, YAP and TAZ. Prolonged inhibition of PHDs was necessary to achieve alterations in cellular localization suggesting an indirect HIF-mediated effect. This mechanism might be extended to other transcription factors and target genes, and may thus represent a novel mechanism of negative regulation of gene expression by PHD inhibition.

The role and related mechanism of YAP on invasion and desmoplastic reaction in pancreatic cancer

Pancreatic cancer (PC), a hypovascular tumor, thrives under hypoxic conditions. Pancreatic stellate cells (PSCs) promote PC progression by secreting soluble factors, but their functions in hypoxia are poorly understood. This study aimed to clarify the effects of hypoxic conditions on the interaction between PC cells and PSCs.We isolated human PSCs from fresh pancreatic ductal adenocarcinomas and analyzed functional differences in PSCs between normoxia (21% O2) and hypoxia (1% O2), including expression of various factors related to tumor–stromal interactions. We particularly analyzed effects on PC invasiveness of an overexpressed molecule—connective tissue growth factor (CTGF)—in PSCs under hypoxic conditions, using RNA interference techniques.Conditioned media from hypoxic PSCs enhanced PC cell invasiveness more intensely than that from normoxic PSCs (P < 0.01). When co-cultured with PSCs, PC cell invasion was more enhanced under hypoxia than under normoxia (P < 0.05). Among various soluble factors, which were related to invasiveness, CTGF was one of the overexpressed molecules in hypoxic PSCs. A higher level of CTGF expression was also found in supernatant of hypoxic PSCs than in supernatant of normoxic PSCs. PC cell invasiveness was reduced by CTGF knockdown in hypoxic PSCs co-cultured with PC cells (P < 0.05).Hypoxia induces PSCs' secretion of CTGF, leading to enhancement of PC invasiveness. CTGF derived from hypoxia-stimulated PSCs may be a new therapeutic target for pancreatic cancer.

A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice.

Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gαq/11 , Gα12/13 and Gαi family without interaction with G protein-coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen-activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular-regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein-dependent signalling in cardiac cells.