Extracellular Matrix Component Genes Research Articles

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

Abstract TMP120: Transcriptomic Analysis Reveals Common Age Independent Immunomodulatory Proteins As A Mode Of Neuroprotection In P2X4R KO Mice After Ischemic Stroke

Background and Purpose: Identification of a new potential drug target protein and their plausible mechanism for stroke treatment is critically needed. We earlier showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute neuroprotection and thus can be potential drug targets to treat ischemic stroke. However, potential mechanisms remain unknown. Therefore, in this study, we employed RNA-seq technology to identify the gene expression profiles, pathways analysis and qPCR validation of differentially expressed genes (DEGs). This analysis will identify role of those DEGs in certain biological processes responsible for P2X4R dependent neuroprotection after stroke. Methods: We subjected young (8-12 weeks-old n= 4/group), and aged (12-18-month-old; n=8/group) male and female Global P2X4 KO and littermate WT mice to right middle cerebral artery occlusion MCAo for 60 min followed by 3 day of reperfusion. After 3 days, mice were sacrificed and prefrontal cortex tissue was isolated to extract total RNA using Trizol and used for RNA-seq sample preparations as well as for validation by Nanostring mediated qPCR technique. DESeq2 and Gene Ontology (GO) and /or Ingenuity Pathway Analysis (IPA) were used to identify mRNA transcript expression profiles and biological pathway. qPCR was analyzed with nSolver Data Analysis Support system. Results: We found 2246 DEGs in P2X4R KO vs WT tissue after stroke. Out of these DEGs 1920 gene were downregulated and 325 genes were upregulated in KO. GO/IPA analysis of top 300 DEGs suggests an enrichment of ion channel transport system, inflammation and extracellular matrix component genes. QPCR validation of top 30 DEGs revealed down regulation of two common age independent genes: Lnterleukin-6 ( IL-6) , an inflammatory cytokine and Cytotoxic T Lymphocyte-Associated Protein 2 alpha ( Ctla2a ), an immunosuppressive factor KO group. Conclusion: This data suggests P2X4R mediated neuroprotection after stroke is brought by attenuation of immune modulatory pathways in both young and aged mice of both sexes. Future studies will delineate the detailed role of IL-6 and Ctla2a in P2X4R mediated neuroprotection mechanisms after stroke.

Transcriptome analysis of collagen VI-related muscular dystrophy muscle biopsies.

ObjectiveTo define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI‐related muscular dystrophy (COL6‐RD).MethodsCOL6‐RD patient muscle biopsies were stratified into three groups based on the overall level of pathologic severity considering degrees of fibrosis, muscle fiber atrophy, and fatty replacement of muscle tissue. Using microarray and RNA‐Seq, we then performed global gene expression profiling on the same muscle biopsies and compared their transcriptome with age‐ and sex‐matched controls.ResultsCOL6‐RD muscle biopsy transcriptomes as a group revealed prominent upregulation of muscle extracellular matrix component genes and the downregulation of skeletal muscle and mitochondrion‐specific genes. Upregulation of the TGFβ pathway was the most conspicuous change across all biopsies and was fully evident even in the mildest/earliest histological group. There was no difference in the overall transcriptional signature between the different histologic groups but polyserial analysis identified relative changes along with COL6‐RD histological severity.InterpretationOverall, our study establishes the prominent dysregulation of extracellular matrix genes, TGFβ signaling, and its downstream cellular pathways at the transcriptomic level in COL6‐RD muscle.

Abstract MP21: Transcriptomic Modulation Of Second Heart Field-derived Smooth Muscle Cells In Angiotensin Ii-infused Mice Promotes Aortopathy

Objective: Smooth muscle cells (SMCs) in the ascending aorta are derived from both the cardiac neural crest and second heart field (SHF). The importance of cardiac neural crest-derived SMCs on development of thoracic aortic aneurysms has been reported, while functional roles of SHF-derived SMCs are controversial. The aim of this study was to profile the transcriptome of SHF-derived SMCs in ascending aortas of angiotensin II (AngII)-infused mice by single cell RNA sequencing. Methods and Results: Female ROSA26 mT/mG mice were bred to male mice expressing Cre under the control of the Mef2c promoter (Mef2c Cre). Angiotensin II (AngII, 1,000 ng/kg/day) was infused subcutaneously into Mef2c Cre +/- mice and ascending aortas were harvested at day 3 of AngII infusion (n=5). Ascending aortas without AngII infusion were harvested as control (n=4). SHF-derived cells were sorted based on mGFP signal by FACS and single cell sequencing was performed using SHF-derived cells. Single cell RNA sequencing detected mRNA in multiple cell types, and AngII altered mRNA abundance of 3,658 genes in the SMC cluster. Several SMC contractile-related genes, such as Acta2, Tagln and Cnn1 , were increased by AngII compared to control SHF-derived SMCs. Conversely , Klf4 , a proliferative gene, was decreased in SHF-derived SMCs of AngII-infused mice. In addition, AngII increased multiple extracellular matrix component genes, including fibrillin1 and elastin. AngII also upregulated Serpine1 and Fn1 that are important for extracellular matrix organization. Thus, AngII led to transcriptomic modulations in SHF-derived SMCs. Of note, AngII infusion decreased mRNA abundance of Tgfbr2 and Lrp1 , key regulators of extracellular matrix maturation, in SHF-derived SMCs. To further investigate the importance of SHF-derived SMCs, we deleted either Tgfbr2 or Lrp1 in SHF-derived cells. SHF-specific Tgfrb2 deletion was embryonic lethal with peritoneal hemorrhage and outflow tract dilatation. SHF-specific Lrp1 deletion augmented AngII-induced thoracic aortic rupture and dilatations. Conclusion: SHF-derived SMCs play an important role in maintaining aortic integrity, and exhibit transcriptomic modulations by AngII infusion that may contribute to aortopathies.

140 Relaxin Regulates Porcine Adipose Tissue Development by Inhibiting Preadipocyte Number, Stimulating Lipolysis, and Upregulating mRNA Expression of Adipokine, Fatty Acid Metabolism and Extracellular Matrix Genes

Abstract Relaxin (RLN) is best known as a reproductive hormone that remodels cervical connective tissue. However, porcine adipose tissue can secrete and respond to RLN, suggesting locally derived RLN could modulate adipose tissue development and function. To test this hypothesis, primary cultures of porcine subcutaneous adipose tissue stromal-vascular (AT-SV) cells and visceral adipose tissue explants were utilized as model systems to determine the effect of 100 ng/ml exogenous RLN on 1) preadipocyte proliferation and differentiation, 2) the mRNA expression of adipokine, extracellular matrix protein and fatty acid metabolism genes, and 3) lipolytic rate in porcine adipose tissue. AT-SV cells were harvested from 3-day-old neonatal pigs (n=6) and plated in vitro in growth medium containing either vehicle or RLN for cell number assays or were induced to differentiate by treatment with insulin, hydrocortisone, and rosiglitazone for 8 days in the presence or absence of RLN for adipogenesis assays. Total RNA was extracted from differentiating cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for target genes. For lipolysis and cell signaling experiments, visceral adipose tissue was obtained from market weight pigs at harvest, minced into explants, and cultured in serum-free medium in the presence or absence of RLN and cAMP pathway modulators with differences in glycerol release determined following 2-hour treatment. Overall, RLN decreased preadipocyte number 1.27-fold under serum-free conditions (P < 0.05), enhanced adipogenesis based upon 2.4-fold increase in GAPDH activity (P < 0.05) and a 2.2-fold increase in ORO accumulation (P < 0.05) while significantly increasing the mRNA expression of multiple metabolic, adipokine, and extracellular matrix component genes. RLN treatment stimulated glycerol release by 3-fold via the cAMP pathway (P < 0.01). Collectively, these data support the hypothesis that RLN regulates adipose tissue development through stimulating adipogenesis and modulating adipocyte metabolism.

Collagen and soy peptides attenuate contractile loss from UVA damage and enhance the antioxidant capacity of dermal fibroblasts.

Wrinkles and extracellular matrix (ECM) loss are common signs of skin aging and are thought to be the result of damage caused by reactive oxygen species (ROS); ROS induces an imbalance between ECM degradation and production. In this study, we evaluate soy peptides (SP) and collagen peptides (CP), alone and in combination, for their ability to inhibit ROS formation and increase ECM gene expression in order to ameliorate the signs of skin aging. Using tert-Butyl hydroperoxide (t-BuOOH)-treated dermal fibroblasts, we explored the potential of CP and SP to inhibit ROS formation by flow cytometry, as well as their effect on ECM component genes by real-time quantitative PCR. In addition, we examined the effect of CP and SP on UVA irradiated fibroblasts in a 3D collagen lattice model that measured contractility. The results showed that the combination of CP and SP synergistically reduces ROS formation. This combination also increased expression of collagen I, collagen II, elastin, and fibronectin in t-BuOOH-treated or untreated dermal fibroblasts. In the UVA-treated 3D collagen lattice model, the results show that CP and SP significantly improved fibroblast contractility when compared to UVA control (P<0.05). In conclusion, CP and SP attenuate the loss of contractility due to UVA damage, inhibit t-BuOOH-induced ROS formation, and improve expression of ECM component genes.

Adhesion G-protein coupled receptor 56 is required for 3T3-L1 adipogenesis.

Obesity-associated conditions represent major global health and financial burdens and understanding processes regulating adipogenesis could lead to novel intervention strategies. This study shows that adhesion G-protein coupled receptor 56 (GPR56) gene transcripts are reduced in abdominal visceral white adipose tissue derived from obese Zucker rats versus lean controls. Immunostaining in 3T3-L1 preadipocytes reveals both mitotic cell restricted surface and low level general expression patterns of Gpr56. Gpr56 transcripts are differentially expressed in 3T3-L1 cells during adipogenesis. Transient knockdown (KD) of Gpr56 in 3T3-L1 cells dramatically inhibits differentiation through reducing the accumulation of both neutral cellular lipids (56%) and production of established adipogenesis Pparγ 2 (60-80%), C/ebpα (40-78%) mediator, and Ap2 (56-80%) marker proteins. Furthermore, genome editing of Gpr56 in 3T3-L1 cells created CW2.2.4 and RM4.2.5.5 clones (Gpr56 -/- cells) with compound heterozygous deletion frameshift mutations which abolish adipogenesis. Genome edited cells have sustained levels of the adipogenesis inhibitor β-catenin, reduced proliferation, reduced adhesion, altered profiles, and or abundance of extracellular matrix component gene transcripts for fibronectin, types I, III, and IV collagens and loss of actin stress fibers. β-catenin KD alone is insufficient to restore adipogenesis in Gpr56 -/- cells. Together these data show that Gpr56 is required for adipogenesis in 3T3-L1 cells. This report is the first demonstration that Gpr56 participates in regulation of the adipogenesis developmental program. Modulation of the levels of this protein and/or its biological activity may represent a novel target for development of therapeutic agents for the treatment of obesity.

Paracrine effects of mesenchymal stem cells on the activation of keratocytes

AimsThe purpose of this study is to investigate the impact of mesenchymal stem cell (MSC)-derived soluble factors on the function of keratocytes, with a particular focus on the processes involved...

Plasminogen Activator Inhibitor Type I Controls Cardiomyocyte Transforming Growth Factor-β and Cardiac Fibrosis.

Fibrosis is the pathological consequence of stress-induced tissue remodeling and matrix accumulation. Increased levels of plasminogen activator inhibitor type I (PAI-1) have been shown to promote fibrosis in multiple organ systems. Paradoxically, homozygous genetic deficiency of PAI-1 is associated with spontaneous age-dependent, cardiac-selective fibrosis in mice. We have identified a novel PAI-1-dependent mechanism that regulates cardiomyocyte-derived fibrogenic signals and cardiac transcriptional pathways during injury. Cardiac fibrosis in subjects with homozygous mutation in SERPINE-1 was evaluated with late gadolinium-enhanced cardiac magnetic resonance imaging. A murine cardiac injury model was performed by subcutaneous infusion of either saline or Angiotensin II by osmotic minipumps. We evaluated blood pressure, cardiac function (by echocardiography), fibrosis (with Masson Trichrome staining), and apoptosis (with TUNEL staining), and we performed transcriptome analysis (with RNA sequencing). We further evaluated fibrotic signaling in isolated murine primary ventricular myocytes. Cardiac fibrosis was detected in 2 otherwise healthy humans with complete PAI-1 deficiency because of a homozygous frameshift mutation in SERPINE-1. In addition to its suppressive role during spontaneous cardiac fibrosis in multiple species, we hypothesized that PAI-1 also regulates fibrosis during cardiac injury. Treatment of young PAI-1-/- mice with Angiotensin II induced extensive hypertrophy and fibrotic cardiomyopathy, with increased cardiac apoptosis and both reactive and replacement fibrosis. Although Angiotensin II-induced hypertension was blunted in PAI-1-/- mice, cardiac hypertrophy was accelerated. Furthermore, ventricular myocytes were found to be an important source of cardiac transforming growth factor-β (TGF-β) and PAI-1 regulated TGF-β synthesis by cardiomyocytes in vitro as well as in vivo during cardiac injury. Transcriptome analysis of ventricular RNA after Angiotensin II treatment confirmed that PAI-1 deficiency significantly enhanced multiple TGF-β signaling elements and transcriptional targets, including genes for extracellular matrix components, mediators of extracellular matrix remodeling, matricellular proteins, and cardiac integrins compared with wild-type mice. PAI-1 is an essential repressor of cardiac fibrosis in mammals. We define a novel cardiomyocyte-specific regulatory mechanism for TGF-β production by PAI-1, which explains the paradoxical effect of PAI-1 deficiency in promoting cardiac-selective fibrosis. Thus, PAI-1 is a molecular switch that controls the cardiac TGF-β axis and its early transcriptional effects that lead to myocardial fibrosis.

Two fluoroquinolones and their combinations with hyaluronan: comparison of effects on canine chondrocyte culture

Fluoroquinolones (FQs) are frequently used for septic arthritis. Increased antibacterial activity has been associated with mammalian cell cytotoxicity that may increase the risk of developing osteoarthritis. This study compared the direct effects of two different FQs, enrofloxacin (Enro) and marbofloxacin (Mar), on normal primary canine chondrocytes and inflammatory-stimulated chondrocytes, in addition to their administration in combination with hyaluronan (HA). Cell viability, cell apoptosis, s-GAG production, and expression patterns of inflammatory, extracellular matrix (ECM) component and protease genes were measured. Enro co-culturing with HA could modify s-GAG synthesis compared with the negative control group. Co-treatment with both FQs and HA significantly decreased cell viability and induced more total apoptotic cell death compared with the negative control and pre-IL-1β-stimulated group. Enro regulated IL-1β-stimulated cells to overexpress IL-1β, TNF, and MMP3, whereas Mar induced upregulation of PTGS2 and NFKB1 and enhanced the expression of ECM component genes HAS1, COL2A1, and ACAN as well as TIMP1 and MMP9. Simultaneous use of HA with Enro can effectively reduce the expression of IL-1β, TNF, and MMP3 in pre-IL-1β-stimulated chondrocytes. These results suggest the beneficial effects of HA in reducing the adverse effects of Enro treatment at the transcriptional level.

Non-human Primate and Rat Cardiac Fibroblasts Show Similar Extracellular Matrix-related and Cellular Adhesion Gene Responses to Substance P

The sensory nerve neuropeptide substance P (SP) regulates cardiac fibrosis in rodents under pressure overload conditions. Interestingly, SP induces transient increased expression of specific genes in isolated rat cardiac fibroblasts, without resultant changes in cell function. This suggests that SP 'primes' fibroblasts, but does not directly activate them. We investigated whether these unusual findings are specific to rodent fibroblasts or are translatable to a larger animal model more closely related to humans. We compared the effects of SP on genes associated with extracellular matrix (ECM) regulation, cell-cell adhesion, cell-matrix adhesion and ECM in cardiac fibroblasts isolated from a non-human primate and Sprague-Dawley rats. We found that rodent and non-human primate cardiac fibroblasts showed similar responses in genes that relate to ECM regulation and cell adhesion in response to SP. There were large discrepancies in ECM component genes, however, this did not result in collagen or laminin synthesis in rat or non-human primate fibroblasts in response to SP. This study further supports the notion that SP serves as a 'primer' for fibroblasts rather than initiating direct effects and suggests that rodent fibroblasts are a suitable model for studying gene and functional responses to SP in the absence of human or non-human primate fibroblasts.

Macrophages are required for adult salamander limb regeneration

The failure to replace damaged body parts in adult mammals results from a muted growth response and fibrotic scarring. Although infiltrating immune cells play a major role in determining the variable outcome of mammalian wound repair, little is known about the modulation of immune cell signaling in efficiently regenerating species such as the salamander, which can regrow complete body structures as adults. Here we present a comprehensive analysis of immune signaling during limb regeneration in axolotl, an aquatic salamander, and reveal a temporally defined requirement for macrophage infiltration in the regenerative process. Although many features of mammalian cytokine/chemokine signaling are retained in the axolotl, they are more dynamically deployed, with simultaneous induction of inflammatory and anti-inflammatory markers within the first 24 h after limb amputation. Systemic macrophage depletion during this period resulted in wound closure but permanent failure of limb regeneration, associated with extensive fibrosis and disregulation of extracellular matrix component gene expression. Full limb regenerative capacity of failed stumps was restored by reamputation once endogenous macrophage populations had been replenished. Promotion of a regeneration-permissive environment by identification of macrophage-derived therapeutic molecules may therefore aid in the regeneration of damaged body parts in adult mammals.

Interleukin-1-mediated effects of normal oral keratinocytes and head and neck squamous carcinoma cells on extracellular matrix related gene expression in fibroblasts

The composition of tumor stroma and the activity of tumor associated fibroblasts are important for tumor growth. Interactions between carcinoma cells and fibroblasts regulate the turnover of extracellular matrix (ECM). Here, the in vitro effects of oral squamous cell carcinoma (SCC) cells (UT-SCC-30 and UT-SCC-87) on fibroblast expression of genes for ECM components and connective tissue growth factor (CTGF/CCN2), were compared to those of normal oral keratinocytes (NOK). Cocultures with fibroblasts in collagen gels and keratinocytes with the two cell types separated by a semi permeable membrane were used, and relative gene expression was measured with real-time PCR. All investigated genes were regulated by NOK and the SCCs. The downregulation of pro-collagens α1(I) and α1(III) was more pronounced in cocultures with NOK, while the expression of CCN2 and fibronectin was downregulated by both NOK and the SCCs to a similar extent. UT-SCC-87, but not UT-SCC-30, secreted significantly more IL-1α than NOK. A recombinant interleukin-1 receptor antagonist reversed many of the observed effects on fibroblast gene expression suggesting involvement of IL-1 in cocultures with NOK as well as with SCCs. The observed differential effects on fibroblast gene expression suggest that NOK are more antifibrotic compared to UT-SCC-30 and UT-SCC-87. These findings may contribute to a better understanding of the mechanisms behind ECM turnover in tumors.

The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro

Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.

Emodin Suppression of Ocular Surface Inflammatory Reaction

To determine whether a Chinese herbal medicine component, emodin, suppresses inflammatory/fibrogenic reaction in cultured subconjunctival fibroblasts and reduces injury-induced increases in ocular surface inflammation in mice. Effects of emodin were measured in human subconjunctival fibroblasts on proliferation and migration with colorimetry and scratch wound assay, respectively. Neovascularization was evaluated using an endothelial cell-fibroblast coculture model. Proinflammatory mediator and extracellular matrix component gene and protein expression was characterized with real-time reverse transcription-polymerase chain reaction, enzyme immunoassay, and immunocytochemistry, respectively. Western blotting and immunohistochemistry evaluated the activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). In a mouse corneal alkali-burn model, the effects of emodin on ocular surface inflammation and fibrosis were evaluated. Emodin suppressed tumor necrosis factor alpha (TNF-alpha)-induced fibroblast migration and fibronectin deposition in vitro. VEGF induced neovascularization but did not affect cell proliferation and collagen type 1 production. Monocyte/macrophage-chemoattractant protein-1 gene and protein expression declined. Emodin inhibited TNF-alpha-induced NF-kappaB p65 and JNK activation but did not affect transforming growth factor beta1-induced Smad2/3 signaling. In vivo, emodin inhibited proinflammatory and fibrogenic reactions. Emodin suppressed in vitro TNF-alpha-induced stimulation of proinflammatory reaction. In a mouse ocular alkali burn model, this herbal component lessened inflammation and scarring. Additional studies are warranted to evaluate the therapeutic potential of emodin in lessening ocular tissue inflammation and resultant fibrosis after injury.

Interaction of angiogenic microvessels with the extracellular matrix

The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues, as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis. During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak matrix metalloproteinase mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM components and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature.

Chromosomal aberrations and gene expression profiles in non-small cell lung cancer

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.

Selective assembly of fibulin‐1 splice variants reveals distinct extracellular matrix networks and novel functions for perlecan/UNC‐52 splice variants

Fibulin-1C and fibulin-1D splice variants have been conserved throughout metazoan evolution and have distinct functions in Caenorhabditis elegans development. Both splice variants are required for the assembly of hemidesmosome-mediated mechanosensory neuron and uterine attachments, although the molecular associations that underlie their distinct functions at these locations are not known. Here, we show that the assembly of fibulin-1C and fibulin-1D splice variants at these anchorages is dependent upon distinct components of the extracellular matrix (ECM): Fibulin-1D assembly at uterine and mechanosensory neurons attachments is dependent upon a perlecan/ UNC-52 splice variant that includes alternately spliced IG8-IG10, whereas the assembly of fibulin-1C at mechanosensory neuron attachments is dependent upon laminin/ EPI-1. These data not only indicate that fibulin-1C and fibulin-1D are components of distinct networks of ECM but also demonstrates a novel function for a major class of perlecan splice variants found in C. elegans and mouse. In addition, we demonstrate that overexpression of another ECM protein, collagen XVIII, can suppress gonad morphogenesis defects associated with loss of fibulin-1C, suggesting that some genetic defects that result in a weakened basement membrane can be compensated by overexpression of genes for ECM components that stabilize basement membranes.

Altered expression of the Smad signalling pathway: implications for COPD pathogenesis

Pulmonary emphysema, as a feature of chronic obstructive pulmonary disease (COPD), is characterised by destruction of alveolar tissue. The present authors previously demonstrated reduced decorin expression in the peribronchial area of COPD patients, reflecting an altered extracellular matrix (ECM) modulation. Decorin transcription is regulated by the transforming growth factor (TGF)-beta-Smad pathway, the key intracellular signal route for initiation of ECM component gene transcription. Whether this pathway is aberrantly expressed in COPD is not known. An immunohistochemical study was performed to compare protein expression of TGF-beta1 and TGF-beta receptors, Smad 2, 3, 4 and 7, and decorin in lung tissue of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages II and IV COPD patients and controls. Epithelial expression of the inhibitory Smad 7 was significantly lower in patients with GOLD stages II and IV than in controls, with other Smad protein expressions being similar in the groups. The expression of TGF-beta1 and TGF-beta receptor type I was significantly lower in stage II patients. Decorin staining of the adventitia and alveolar walls was significantly reduced in COPD stage IV. In conclusion, the transforming growth factor-beta-Smad pathway is aberrantly expressed in chronic obstructive pulmonary disease patients, implying an abnormal tissue repair ultimately resulting in reduced decorin production. The results of the present study contribute to better understanding of the pathogenesis of emphysema and the airway fibrosis observed in chronic obstructive pulmonary disease patients.

The candidate tumor suppressor CST6 alters the gene expression profile of human breast carcinoma cells: Down-regulation of the potent mitogenic, motogenic, and angiogenic factor autotaxin

We recently coined CST6 as a novel candidate tumor suppressor gene for breast cancer. CST6 indeed is expressed in the normal human breast epithelium, but little or not at all in breast carcinomas and breast cancer cell lines. Moreover, ectopic expression of CST6 in human breast cancer cells suppressed cell proliferation, migration, invasion, and orthotopic tumor growth. To obtain insights into the molecular mechanism by which CST6 exhibits its pleiotropic effects on tumor cells, we compared global gene expression profiles in mock- and CST6-transfected human MDA-MB-435S cells. Out of 12,625 transcript species, 61 showed altered expression. These included genes for extracellular matrix components, cytokines, kinases, and phosphatases, as well as several key transcription factors. TaqMan PCR assays were used to confirm the microarray data for 7 out of 11 genes. One down-regulated gene product, secreted autotaxin/lyso-phospholipase D, was of particular interest because its down-regulation by CST6 could explain most of CST6’s effect on the breast cancer cells. This study thus provides the first evidence that CST6 plays a role in the modulation of genes, particularly, genes that are highly relevant to breast cancer progression.