Expression Of ECM Proteins Research Articles

Intravenous delivery of microRNA-133b along with Argonaute-2 enhances spinal cord recovery following cervical contusion in mice

Background contextAcute spinal cord injury (SCI) is a devastating condition for which spine decompression and stabilization of injury remains the only therapy available in the clinical setup. However, fibrous scar formation during the healing process significantly impairs full recovery. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target mRNA(s) and initiating translational repression or mRNA degradation. It has been reported that microRNA-133b (miR133b) is highly expressed in regenerating neurons following a SCI in zebrafish, and lentiviral delivery of miR133b at the time of SCI in mice resulted in improved functional recovery. PurposeThe aim of this study was to investigate whether intravenous delivery of miR133b enhances spinal cord recovery when administered 24 hours following a cervical contusion injury in mice. Study designThis is an experimental animal study of acute SCI, investigating the effect of miR133b on spinal cord recovery by targeting scar lesion formation. The approach involved setting an acute SCI in mice, which was followed 24 hours later by intravenous co-delivery of miR133b and Argonaute 2 (Ago2), a protein involved in miRNA stabilization. Readouts of the impact of this intervention included analysis of RNA and protein expression at the lesion site, in particular with regard to markers of scar tissue formation, and determination of motor function recovery by the grip strength meter task. MethodsC57BL6 female mice between 6 and 8 weeks of age were tested. The injury model employed was a unilateral moderate contusion at the cervical fifth level. Twenty-four hours following the injury, the authors co-delivered miR133b, or scrambled miRNA as negative control, along with Ago2 for 3 consecutive days, one dose per day via tail-vein injection. They first investigated the level of miR133b in the spinal cord and in spinal cord lesion after a single dose of injection. Next, they determined the efficacy of miR133b and/or Ago2 delivery in regulating gene and protein expression at the lesion site. Finally, they established the role of miR133b and/or Ago2 in enhancing forelimb gripping recovery as assessed by the grip strength meter task for 8 weeks post-SCI. ResultsIntravenous delivery of miR133b and/or Ago2 targeted the microenvironment at the lesion site and prevented the increased expression of certain extracellular matrix proteins (ECM), in particular collagen type 1 alpha 1 and tenascin N, which are known to have a key role in scar formation. It also reduced microglia and/or macrophage recruitment to the lesion site. Functional recovery in mice treated with miR133b and/or Ago2 started around 2 weeks postinjury and continued to improve over time, whereas mice in the control group displayed significantly poorer recovery. ConclusionsOur data indicate therapeutic activity of intravenous miR133b and/or Ago2 treatment, possibly via decreasing ECM protein expression and macrophage recruitment at the lesion site, thereby minimizing detrimental fibrous scar formation. Clinical significanceThere is an urgent medical need for better treatments of SCIs. Based on our findings in a preclinical model, the miR133b and/or Ago2 system specifically targets fibrous scar formation, a barrier in neuronal regrowth, by remodeling ECM molecules at the injury site. Prevention of scar formation is critical to improved outcomes of treatment. Of note, delivery of miR133b and/or Ago2 was initiated 24 hours after traumatic impact, thus indicating a fairly long window of opportunity providing more time and flexibility for therapeutic intervention. Intravenous miR133b may become a beneficial therapeutic strategy to treat patients with acute SCI.

Burn-Induced Impairment of Ileal Muscle Contractility Is Associated with Increased Extracellular Matrix Components

IntroductionSevere burns lead to marked impairment of gastrointestinal motility, such as delayed gastric emptying and small and large intestinal ileus. However, the cellular mechanism of these pathologic changes remains largely unknown. MethodsMale Sprague Dawley rats approximately 3 months old and weighing 300–350 g were randomized to either a 60% total body surface area full-thickness scald burn or sham procedure and were sacrificed 24 h after the procedure. Gastric emptying, gastric antrum contractility ileal smooth muscle contractility, and colonic contractility were measured. Muscularis externa was isolated from the ileal segment to prepare smooth muscle protein extracts for Western blot analysis. ResultsCompared with sham controls, the baseline rhythmic contractile activities of the antral, ileal, and colonic smooth muscle strips were impaired in the burned rats. Simultaneously, our data showed that ileal muscularis ECM proteins fibronectin and laminin were significantly up-regulated in burned rats compared with sham rats. TGF-β signaling is an important stimulating factor for ECM protein expression. Our results revealed that TGF-β signaling was activated in the ileal muscle of burned rats evidenced by the activation of Smad2/3 expression and phosphorylation. In addition, the total and phosphorylated AKT, which is an important downstream factor of ECM signaling in smooth muscle cells, was also up-regulated in burned rats’ ileal muscle. Notably, these changes were not seen in the colonic or gastric tissues. ConclusionDeposition of fibrosis-related proteins after severe burn is contributors to decreased small intestinal motility.

Adipose-derived stem cell-conditioned medium protects fibroblasts at different senescent degrees from UVB irradiation damages.

Adipose-derived stem cells (ADSCs) and their derivatives have aroused intense interest in fields of dermatological and aesthetic medicine. As a major component detected in ADSCs secretome, platelet-derived growth factor AA (PDGF-AA) has been reported mediating extracellular matrix deposition and remodeling, thus might contribute to its anti-aging effect. On the basis of establishing an experimental model that simulate actual skin aging by exposing HDFs to both intrinsic and extrinsic aging factors, we pretreated human dermal fibroblasts (HDFs) with ADSC-conditioned medium (ADSC-CM) before being irradiated, aiming at exploring preventive effects of ADSCs secretome against aging damages. 48h after irradiation, we detected cellular proliferation; β-galactosidase stain; mRNA expressions of MMP-1, MMP-9, and TIMP-1; and protein expressions of collagen I, collagen III, and elastin. Moreover, we detected related protein expression of PI3K/Akt signal pathway, which can be activated by PDGF-AA and was newly found to promote extracellular matrix protein synthesis. Concentration of PDGF-AA in the prepared ADSC-CM decreased over time and maintained excellent bioactivity at low temperature until the 11th week. ADSC-CM pretreatment can slightly or significantly improve cellular proliferative activity and reduce cellular senescence in irradiated HDFs. Besides, ADSC-CM pretreatment increased collagen I, collagen III, elastin, and TIMP-1 expressions but decreased MMP-1 and MMP-9 expressions both in irradiated and nonirradiated HDFs. ADSC-CM pretreatment significantly increased pAkt protein expression, and ECM protein expression greatly decreased in case of LY294002 application. The results were similar in three generations of HDFs, yet varied with different degrees. Generally, ADSC-CM we prepared demonstrates a certain degree of positive role in preventing HDFs from intrinsic and extrinsic aging damages and that PDGF-AA may contribute to making it become effective with some other components in ADSC-CM.

Induction of Fibrogenic Phenotype in Human Mesenchymal Stem Cells by Connective Tissue Growth Factor in a Hydrogel Model of Soft Connective Tissue.

Scar formation is the typical endpoint of wound healing in adult mammalian tissues. An overactive or prolonged fibrogenic response following injury leads to excessive deposition of fibrotic proteins that promote tissue contraction and scar formation. Although well-defined in the dermal tissue, the progression of fibrosis is less explored in other connective tissues, such as the vocal fold. To establish a physiologically relevant 3D model of loose connective tissue fibrosis, we have developed a synthetic extracellular matrix using hyaluronic acid (HA) and peptidic building blocks carrying complementary functional groups. The resultant network was cell adhesive and protease degradable, exhibiting viscoelastic properties similar to the human vocal fold. Human mesenchymal stem cells (hMSCs) were encapsulated in the HA matrix as single cells or multicellular aggregates and cultured in pro-fibrotic media containing connective tissue growth factor (CTGF) for up to 21 days. hMSCs treated with CTGF-supplemented media exhibited an increased expression of fibrogenic markers and ECM proteins associated with scarring. Incorporation of α-smooth muscle actin into F-actin stress fibers was also observed. Furthermore, CTGF treatment increased the migratory capacity of hMSCs as compared to the CTGF-free control groups, indicative of the development of a myofibroblast phenotype. Addition of an inhibitor of the mitogen-activated protein kinase (MAPK) pathway attenuated cellular expression of fibrotic markers and related ECM proteins. Overall, this study demonstrates that CTGF promotes the development of a fibrogenic phenotype in hMSCs encapsulated within an HA matrix and that the MAPK pathway is a potential target for future therapeutic endeavors towards limiting scar formation in loose connective tissues.

High amplitude stretching of ATII cells and fibroblasts results in profibrotic effects

Background: Inappropriate mechanical forces act on alveolar epithelial cells during mechanical ventilation e.g. in ARDS and possibly in patients with pulmonary fibrosis. These forces can cause lung injury and may contribute to the development or aggravation of pulmonary fibrosis. Aim of the study: We investigated the hypothesis that high amplitude mechanical stretching of alveolar type II (ATII) cells and lung fibroblasts promotes profibrotic processes. Material and Methods: ATII cells and fibroblasts were stretched on elastic membranes using a pattern of higher amplitudes (“unphysiological”). The production of profibrotic cytokines and extra cellular matrix (ECM) proteins were investigated in supernatants. In addition, we determined the expression of relevant microRNAs (miRNA) and the process of epithelial-mesenchymal transition (EMT) in ATII cells. Results: Unphysiological stretch of ATII cells led to increased release of TGF-β1 into supernatants. We also found elevated protein levels of collagen I and IV in supernatants of stretched cells. By contrast, stretching of fibroblasts changed neither the expression of fibrosis-modulating factors nor ECM-proteins. However, fibroblasts significantly withstood stretch-induced cell injury and seemed to have a survival benefit. Further, stretched ATII cells exhibited a higher expression of miRNAs (miR-15b, miR-25, let-7d) relevant to EMT. The process of EMT, which is characterized by an increase of vimentin and a decrease of cytokeratin expression, was significantly accelerated due to stretching of ATII cells. Conclusion: These data provide evidence that unphysiological mechanical stretching of lung cells induced several profibrotic effects and accelerated EMT, which may have critical implications in terms of development or aggravation of pulmonary fibrosis in the clinical context.

Nimbolide ameliorates unilateral ureteral obstruction-induced renal fibrosis by inhibition of TGF-β and EMT/Slug signalling

Chronic kidney disease (CKD) involves interstitial fibrosis as an underlying pathological process associated with compromised renal function irrespective of etiological cause of the injury. The transforming growth factor-β (TGF-β) plays a pivotal role in progression of renal fibrosis. TGF-β transduces its downstream signalling by phosphorylation of smad2/3 and also regulates epithelial-mesenchymal-transition (EMT), a program centrally involved in activation of fibroblasts. Renal fibrosis was induced in Swiss albino mice by unilateral ureteral obstruction of animals. Kidney tissues were evaluated for fibrotic protein expression by western blot and immunohistochemistry. The administration of nimbolide (NB) to UUO animals reduced the oxidative stress, expression of ECM proteins, TGF-β, p-smad and EMT program. Further, NB administration also improved histoarchitecture of obstructed kidney and reduced the collagen deposition in kidney. Our results provided compelling evidence to support antifibrotic activity of NB by reduction in oxidative stress, TGF-β, and EMT program in fibrotic kidney. The administration of NB in animals blunted the UUO-induced renal injury, inflammation and reduced fibrogenesis in obstructed kidney.

Avian Primordial Germ Cells Contribute to and Interact With the Extracellular Matrix During Early Migration.

During early avian development, primordial germ cells (PGC) are highly migratory, moving from the central area pellucida of the blastoderm to the anterior extra-embryonic germinal crescent. The PGCs soon move into the forming blood vessels by intravasation and travel in the circulatory system to the genital ridges where they participate in the organogenesis of the gonads. This complex cellular migration takes place in close association with a nascent extracellular matrix that matures in a precise spatio-temporal pattern. We first compiled a list of quail matrisome genes by bioinformatic screening of human matrisome orthologs. Next, we used single cell RNA-seq analysis (scRNAseq) to determine that PGCs express numerous ECM and ECM-associated genes in early embryos. The expression of select ECM transcripts and proteins in PGCs were verified by fluorescent in situ hybridization (FISH) and immunofluorescence (IF). Live imaging of transgenic quail embryos injected with fluorescent antibodies against fibronectin and laminin, showed that germinal crescent PGCs display rapid shape changes and morphological properties such as blebbing and filopodia while surrounded by, or in close contact with, an ECM fibril meshwork that is itself in constant motion. Injection of anti-β1 integrin CSAT antibodies resulted in a reduction of mature fibronectin and laminin fibril meshwork in the germinal crescent at HH4-5 but did not alter the active motility of the PGCs or their ability to populate the germinal crescent. These results suggest that integrin β1 receptors are important, but not required, for PGCs to successfully migrate during embryonic development, but instead play a vital role in ECM fibrillogenesis and assembly.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Effect of phlorotannins on myofibroblast differentiation and ECM protein expression in transforming growth factor β1‑induced nasal polyp‑derived fibroblasts.

Phlorotannins (PTNs), a group of phenolic compounds from seaweeds, have diverse bioactivities. However, there has been no report on their antifibrotic effects during nasal polyp (NP) formation. In the present study, the effect of PTNs on transforming growth factor (TGF)‑β1‑induced profibrotic responses in nasal polyp‑derived fibroblasts (NPDFs) were determined and the relevant signaling pathways were investigated. The expression levels of collagen type‑1(Col‑1) and fibronectin in NP tissues were measured by western blot analysis and immunohistochemistry. The NPDFs were treated with TGF‑β1 (1ng/ml) in the presence or absence of PTNs (5‑30µg/ml). The expression levels of α‑smooth muscle actin (α‑SMA), Col‑1, fibronectin, and phosphorylated‑small mothers against decapentaplegic (Smad)2/3 in NPDFs were measured by western blot analysis. The contractile activity of the NPDFs was determined by a collagen gel contraction assay. Col‑1 and fibronectin proteins were found to be expressed in NP tissues. PTNs had no significant cytotoxic effect on TGF‑β1‑induced NPDFs. TGF‑β1 induced the expression α‑SMA, Col‑1 and fibronectin, and stimulated fibroblast‑mediated contraction of collagen gel. However, pre‑treatment with PTNs inhibited the expression of these proteins. The inhibitory effects were mediated through the suppression of Smad2/3 signaling pathways in TGF‑β1‑induced NPDFs. These resulted suggested that PTNs may be important in inhibiting myofibroblast differentiation and extracellular matrix protein accumulation in NP formation through the Smad2/3 signaling pathway.

Abstract 627: Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA)

Abstract The use of oligosaccharides as active pharmaceutical ingredients lags behind that of other biological molecules such as proteins and nucleic acids. However, there is now a growing understanding of the complex biological functions of saccharide structures. This has resulted in an increased interest in exploring the pharmaceutical applications of both oligosaccharides and glycoconjugates. Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late stage disease and are often unable to tolerate drug combination regimens due to the associated toxicity. Here we report a novel oligosaccharide drug candidate, RiXOVA (G-blocks). The term “G-blocks” refers to highly defined and specialized short oligomers comprising α-L-guluronate residues derived from alginates extracted from brown seaweed. The G-block oligomers are well tolerated at i.v./i.p. doses >100mg/kg in mice. These oligomers have previously demonstrated the ability to alter matrices of biological macromolecules including mucins, giving rise to the hypothesis that they may have similar effects on the dense desmoplastic tissue within tumours. We here describe the anti-tumour properties of RiXOVA in different PDAC mouse models. Initial efficacy studies were performed in a xenograft model using the CAPAN-2 cells. RiXOVA was found to be non-toxic and non-immunogenic (I.P, 25mg/kg Q3D10 ) in mice. Relative to the start of dosing, the tumour growth in RiXOVA treated mice was 160% +/-30 (n=10) compared to 240% +/- 61 (n=10, p value=0.001) in the vehicle treated mice. Further testing was carried out in a Genetically Engineered Mouse-Derived Allograft (GEDA). The GEDA model recapitulates the dense desmoplastic stroma of the original donor (KPC mouse), unlike cell line-based allografts. KPC (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-cre) mouse tumour fragments (2 donors) were implanted subcutaneously in the flank of recipient LSL-Trp53R172H; Pdx1-cre (PC), immunocompetent mice. Relative to the start of dosing, the tumour growth in the RiXOVA treated group (25 mg/kg TIW) was 260% +/- 160 (n=10), compared to 540% +/- 210 (n=8) for the Vehicle (p = 0.001) and 130 % +/- 57 (n=9) for the combination of RiXOVA + gemcitabine (100 mg/kg BIW p <0.001) compared to the vehicle. Ex vivo analysis of tumour tissue revealed changes in the expression and distribution of ECM proteins. Collectively, these results indicate RiXOVA to be a non-toxic oligosaccharide, that acts at the level of the tumour microenviroment to inhibit tumour growth. Whilst these results are in a preliminary stage they indicate G-block oligomers may represent a novel treatment modality in PDAC. Citation Format: Shalini V. Rao, Tonje S. Steigedal, Aarthi Gopinathan, Synnøve N. Magnussen, Sabina S. Strand, Duncan Jodrell, Fran Richards, Kurt I. Draget, Catherine T. Nordgård. Antitumor effect of an oligosaccharide API in a genetically engineered mouse-derived allograft (GEDA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 627.

Endoglin haploinsufficiency is associated with differential regulation of extracellular matrix production during skin fibrosis and cartilage repair in mice.

Transforming growth factor (TGF)-β is a multifunctional growth factor with potent pro-fibrotic effects. Endoglin is a TGF-β co-receptor that strongly regulates TGF-β signaling in a variety of cell types. Although aberrant regulation of TGF-β signaling is known to play a key role in fibrotic diseases such as scleroderma and impaired cartilage repair, the significance of endoglin function in regulating these processes is poorly understood. Here we examined whether endoglin haploinsufficiency regulates extracellular (ECM) protein expression and fibrotic responses during bleomycin induced skin fibrosis and surgically induced osteoarthritis, using endoglin-heterozygous (Eng+/-) mice and wild-type (Eng+/+) littermates. Skin fibrosis was induced by injecting mice intradermally with bleomycin or vehicle. Osteoarthritis was induced surgically by destabilization of medial meniscus. Dermal thickness, cartilage integrity and ECM protein expression were then determined. Eng+/- mice subjected to bleomycin challenge show a marked decrease in dermal thickness (P < 0.005) and reduced collagen content and decreased collagen I, fibronectin, alpha-smooth muscle actin levels as compared to Eng+/+ mice, both under basal and bleomycin treated conditions. Eng+/- mice undergoing surgically induced osteoarthritis show no differences in the degree of cartilage degradation, as compared to Eng+/+ mice, although chondrocytes isolated from Eng+/- display markedly enhanced collagen II levels. Our findings suggest that endoglin haploinsufficiency in mice ameliorates bleomycin-induced skin fibrosis suggesting that endoglin represents a pro-fibrotic factor in the mouse skin. However, endoglin haploinsufficiency does not protect these mice from surgically indicedcartilage degradation, demonstrating differential regulation of endoglin action during skin and cartilage repair.

Regulation of fibrotic changes by the synergistic effects of cytokines, dimensionality and matrix: Towards the development of an in vitro human dermal hypertrophic scar model

Current therapeutic strategies to reduce scarring in full thickness skin defect offer limited success due to poor understanding of scar tissue formation and the underlying signaling pathways. There is an urgent need to develop human cell based in vitro scar tissue models as animal testing is associated with ethical and logistic complications and inter-species variations. Pro-inflammatory cytokines play critical role in regulating scar development through complex interplay and interaction with the ECM and corresponding signaling pathways. In this context, we assessed the responses of cultured fibroblasts with respect to their differentiation into myofibroblasts using optimised cytokines (TGF-β1, IL-6 and IL-8) for scar formation in 2D (tissue culture plate, collagen type I coated plate) vs 3D collagen type I gel based constructs. We attempted to deduce the role of dimensionality of cell culture matrix in modulating differentiation, function and phenotype of cultured fibroblasts. Validation of the developed model showed similarity to etiology and pathophysiology of in vivo hypertrophic scar with respect to several features: 1) transition of fibroblasts to myofibroblasts with convincing expression of α-SMA stress fibers; 2) contraction; 3) excessive collagen and fibronectin secretion; 4) expression of fibrotic ECM proteins (SPARC and Tenascin); 5) low MMP secretion. Most importantly, we elucidated the involvement of TGF-β/SMAD and Wnt/β-catenin pathways in developing in vitro dermal scar. Hence, this relatively simple in vitro human scar tissue equivalent may serve as an alternative for testing and designing of novel therapeutics and help in extending our understanding of the complex interplay of cytokines and related dermal scar specific signaling. Statement of SignificanceScarring of the skin affects almost millions of people per year in the developed world alone, nevertheless the complex pathophysiology and the precise signaling mechanisms responsible for this phenomenon of skin scarring are still unknown. A number of anti-scar drugs are being developed and being tested on animals and monolayer models. However, testing the efficacy of these drugs on lab based 3D in vitro models may prove extremely useful in recapitulating the 3D microenvironment of the native scar tissue. In that context in this study we have demonstrated the development of 3D in vitro dermal scar model, by optimizing a constellation of factors, such as combination of cytokines (TGF-β1,IL-6,IL-8) and cellular dimensionality in inducing the differentiation of dermal fibroblasts to myofibroblasts. This in vitro scar model was successful in replicating hallmark features of hypertrophic scar such as excessive synthesis of fibrotic extracellular matrix, perturbed matrix homeostasis, contraction, diminished MMP synthesis. The study also highlighted significant involvement of TGF-β/SMAD and Wnt/β-catenin signaling pathways in in vitro scar formation.

A model of human lung fibrogenesis for the assessment of anti-fibrotic strategies in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited therapeutic options. KCa3.1 ion channels play a critical role in TGFβ1-dependent pro-fibrotic responses in human lung myofibroblasts. We aimed to develop a human lung parenchymal model of fibrogenesis and test the efficacy of the selective KCa3.1 blocker senicapoc. 2 mm3 pieces of human lung parenchyma were cultured for 7 days in DMEM ± TGFβ1 (10 ng/ml) and pro-fibrotic pathways examined by RT-PCR, immunohistochemistry and collagen secretion. Following 7 days of culture with TGFβ1, 41 IPF- and fibrosis-associated genes were significantly upregulated. Immunohistochemical staining demonstrated increased expression of ECM proteins and fibroblast-specific protein after TGFβ1-stimulation. Collagen secretion was significantly increased following TGFβ1-stimulation. These pro-fibrotic responses were attenuated by senicapoc, but not by dexamethasone. This 7 day ex vivo model of human lung fibrogenesis recapitulates pro-fibrotic events evident in IPF and is sensitive to KCa3.1 channel inhibition. By maintaining the complex cell-cell and cell-matrix interactions of human tissue, and removing cross-species heterogeneity, this model may better predict drug efficacy in clinical trials and accelerate drug development in IPF. KCa3.1 channels are a promising target for the treatment of IPF.

MiR-328 prevents renal fibrogenesis by directly targeting TGF-β2.

MicroRNAs have been shown to be crucial in the post-transcriptional regulation of various cellular biological functions. They also play important roles in various human diseases. In renal fibrosis, aberrant miRNA expression is observed. However, little is known about the roles of miR-328 in renal fibrogenesis and its regulating mechanisms. In the present study, we investigated the miR-328 expression profile in TGF-β1-induced renal fibrogenesis cell model using qRT-PCR. The results showed that TGF-β1 could induce EMT expression and fibrogenesis and significantly down-regulated the expression of miR-328. Through bioinformatical analysis, we identified that TGF-β2 was a direct target gene of miR-328. Luciferase reporter assay was conducted to further confirm this finding. The results also demonstrated that miR-328 mimics transfection could significantly upregulate miR-328 expression. Furthermore, upregulation of miR-328 could further repress the expression of TGF-β2 and ECM proteins. In conclusion, this study demonstrated that miR-328 could prevent renal fibrogenesis by directly targeting TGF-β2. Our findings suggested that elevated renal miR-328 levels might be a novel therapeutic strategy for treating renal fibrosis (Fig. 4, Ref. 36).

Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells.

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+ entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+ channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+ channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+ channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

Alpha-lipoic acid ameliorates the epithelial mesenchymal transition induced by unilateral ureteral obstruction in mice

The epithelial-to-mesenchymal transition (EMT) is one of mechanisms that induce renal interstitial fibrosis. Understanding EMT in renal fibrosis has important therapeutic implications for patients with kidney disease. Alpha-lipoic acid (ALA) is a natural compound with antioxidant properties. Studies for ALA are performed in acute kidney injury with renal tubular apoptosis, renal inflammation, and oxidative stress. We investigated the effects of ALA on EMT-mediated renal interstitial fibrosis in mice with unilateral ureteral obstruction (UUO). UUO mice developed severe tubular atrophy and tubulointerstitial fibrosis, with a robust EMT response and ECM deposition after 7 postoperative days. In contrast, ALA-treated UUO mice showed only moderate injury and minimal fibrosis and also larger reductions in the expression of ECM proteins, inflammatory factors, and EMT markers. ALA was shown to be involved in the suppression of infiltrating macrophages associated with EMT and the progression of interstitial fibrosis. It also lessened the destruction of the tubular basement membrane, by reducing the expression of matrix metalloproteinases. This is the first study to show that ALA modulates EMT in a UUO mouse model. Our results suggest that ALA merits further exploration as a therapeutic agent in the prevention and treatment of chronic kidney disease.

Highlights of This Issue

Multigene assays have been incorporated into the management of patients with node-negative hormone receptor-positive breast cancer for determining adjuvant treatment strategies. To compare prognostic accuracy of two multigene assays, Sestak and colleagues examined cross-stratification between Breast Cancer Index (BCI) and the 21-gene recurrence score (RS) in the TransATAC study. The findings show that BCI demonstrates increased prognostic accuracy versus RS. BCI restratified women with low and intermediate risk by RS into clinically relevant risk groups with significantly different risks of distant recurrence. These findings indicate that BCI may have potential impact to improve individualized risk stratification for patients with early-stage breast cancer.Dennison and colleagues evaluated the associative effects of breast cancer with the tumor microenvironment and its influence on tumor behavior. The reactive subtype of breast cancer, identified by reverse-phase protein arrays to have high expression of ECM proteins, was demonstrated to indicate a favorable outcome. The lowest risk tumors, typically ER+ER2− breast cancer, were more likely to have high expression of stromal proteins, high intratumoral stromal volume, and increased cell density within the stromal compartment.Antibody–drug conjugates (ADC) have become a promising anticancer agent. Ogitani and colleagues generated and evaluated a novel HER2-targeting ADC called DS-8201a, with a DNA topoisomerase I inhibitor. It showed potent antitumor activity against T-DM1-insensitive tumors and HER2 low-expressing tumors due to the different mechanism payload from T-DM1 and higher a drug-to-antibody ratio than T-DM1. Moreover, favorable pharmacokinetic and safety profiles in monkeys were observed. These data suggest that DS-8201a is an attractive and promising HER2-targeting ADC, which provides an additional treatment option for current HER2-targeting therapy.Antibody–drug conjugates directed at specific antigens preferentially expressed on cancer cells are gaining traction in the clinic. Here, Almhanna and colleagues perform the first-in-human phase 1 study investigating TAK-264 targeting guanylyl cyclase, a transmembrane cell-surface receptor expressed in many gastrointestinal cancers. The results demonstrate a manageable safety profile at the maximum tolerated dose, preliminary evidence of antitumor activity, and early signs of clinical benefit in patients with pancreatic, esophageal, and gastric carcinoma.

Hydrogel-based microfluidics for vascular tissue engineering

Abstract In this work, we have explored 3-D co-culture of vasculogenic cells within a synthetically modified fibrin hydrogel. Fibrinogen was covalently linked with PEG-NHS in order to improve its degradability resistance and physico-optical properties. We have studied influences of the degree of protein PEGylation and the concentration of enzyme thrombin used for the gel preparation on cellular responses. Scanning electron microscopy analysis of prepared gels revealed that the degree of PEGylation and the concentration of thrombin strongly influenced microstructural characteristics of the protein hydrogel. Human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem cells (hASCs), used as vasculogenic co-culture, could grow in 5:1 PEGylated fibrin gels prepared using 1:0.2 protein to thrombin ratio. This gel formulation supported hASCs and HUVECs spreading and the formation of cell extensions and cell-to-cell contacts. Expression of specific ECM proteins and vasculogenic process inherent cellular enzymatic activity were investigated by immunofluorescent staining, gelatin zymography, western blot and RT-PCR analysis. After evaluation of the optimal gel composition and PEGylation ratio, the hydrogel was utilized for investigation of vascular tube formation within a perfusable microfluidic system. The morphological development of this co-culture within a perfused hydrogel over 12 days led to the formation of interconnected HUVEC-hASC network. The demonstrated PEGylated fibrin microfluidic approach can be used for incorporating other cell types, thus representing a unique experimental platform for basic vascular tissue engineering and drug screening applications.

Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis

Coronary atherosclerosis complicated by plaque disruption and thrombosis is a critical event in myocardial infarction and stroke, the major causes of cardiovascular death. In atherogenesis, macrophages (MAC) and smooth muscle cells (SMC) are key actors; they synthesize matrix components and numerous factors involved in the process. Here, we design experiments to investigate whether SMC–MAC communication induces changes in ECM protein composition and/or neo-angiogenesis. Cell to cell communication was achieved using trans-well chambers, where SMCs were grown in the upper chamber and differentiated MAC in the bottom chamber for 24 or 72h. We found that cross-talk between MAC and SMC during co-culture: (i) significantly decreased the expression of ECM proteins (collagen I, III, elastin) in SMC; (ii) increased the expression and activity of metalloprotease MMP-9 and expression of collagenase MMP-1, in both MAC and SMC; (iii) augmented the secretion of soluble VEGF in the conditioned media of cell co-culture and VEGF gene expression in both cell types, compared with control cells. Moreover, the conditioned media collected from MAC–SMC co-culture promoted endothelial cell tube formation in Matrigel, signifying an increased angiogenic effect. In addition, the MAC–SMC communication led to an increase in inflammatory IL-1β and TLR-2, which could be responsible for cellular signaling. In conclusion, MAC–SMC communication affects factors and molecules that could alter ECM composition and neo-angiogenesis, features that could directly dictate the progression of atheroma towards the vulnerable plaque. Targeting the MAC–SMC cross-talk may represent a novel therapeutic strategy to slow-down or retard the plaque progression.

Abstract 2385: Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HtrA1 and deregulated TGFB signaling

Abstract Dissemination of ovarian cancer is promoted by deregulated cell adhesion that selectively enhances detachment at primary site and attachment and invasion at secondary sites. Modifications in the extracellular matrix composition within tumor microenvironment can facilitate deregulated cell adhesion, migration, and invasion. We aim to understand how tumor-intrinsic changes affect expression and composition of ECM proteins in disseminated ovarian cancer. The serine protease HtrA1 has been shown to inhibit TGFB by targeting TGFB family ligands and receptors for proteolysis. In addition, HtrA1 also degrades ECM proteins, such as decorin, fibronectin, and collagens. We have shown that HtrA1 is down-regulated in ovarian cancer cells with loss contributing to chemotherapy resistance. We propose that loss of HtrA1 in the ovarian tumor microenvironment enhances TGFB signaling that in turn facilitate compositional changes in ECM proteins from stromal cells and promote cancer dissemination. To characterize compositional changes that occur in ECM proteins as a result of increased TGFB signaling, stable isotope amino acid labeling (SILAC) and LC-MS analysis were used. Mesothelial cells were cultured in heavy or light isotopically labeled media to confluence. Tumor cells expressing HtrA1 (NT) or stably transfected with HtrA1-targeting shRNA (sh1) were added to either heavy or light mesothelial cultures with or without TGFB (10 ng/ml). Cells were lifted by EDTA and the remaining matrix proteins were subjected to LC-MS. The data show that ECM compositional changes in fibronectin, laminin and collagen were up regulated in mesothelial cells co-cultured with sh1 compared to NT cells. All extracted proteins with ≥ 4 fold change were analyzed by Ingenuity Pathway Analysis. Proteins up regulated in co-cultures with HtrA1-deficient cancer cells (sh) were determined to be associated with migration, invasion and tumorogenesis whereas proteins upregulated in co-culture with HtrA1-expressing cancer cells (NT) were associated with protein modifications and ER stress. HtrA1-deficient cells show decreased adhesion to collagen I/III matrix and enhanced adhesion to collagen I/VI matrix. HtrA1-deficient cancer cells show morphology characteristics of epithelial-mesenchymal transition and shows higher motility and invasion through mesothelial cell layer. Finally, in clinical samples, decreased HtrA1 expression in primary tumor was significantly associated with higher incidence of carcinomatosis and nodal metastasis in ovarian cancer. We conclude that loss of HtrA1 in ovarian cancer cells results in compositional changes in ECM within the tumor microenvironment, and these changes may contribute to the dissemination of ovarian cancer. Citation Format: Megan Cooley, Jeremy Chien, Anirban Mitra, Alfonso Baldi, Andras Czirok, Edina Kosa. Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HtrA1 and deregulated TGFB signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2015-2385