Role Of Extracellular Matrix Proteins Research Articles

The Role of Extracellular Matrix Protein 1 (ECM1) in Cardiac Fibrosis

The Role of Extracellular Matrix Protein 1 (ECM1) in Cardiac Fibrosis

Extracellular matrix proteins in healthy and retained placentas, comparing hemochorial and synepitheliochorial placentas

The placenta expresses structural and biologically active proteins. Their synthesis is mainly regulated by genomic or nongenomic signals and modulated by hormones. These protein profiles are altered during different stages of pregnancy. The biological properties of extracellular matrix (ECM) proteins were defined and described in a number of tissues including placenta. These properties enable them to be the main players in the processes of attachment or invasion into the endometrium during initial placenta formation and its timely separation after delivery and detachment. In this review, we focused on the role of ECM proteins during attachment of the placenta to the uterine wall, its timely separation, and the implications of this process on retained or pathologically attached placenta.Although the amount of published information in this area is relatively scant, some of the key proteins and processes are well defined. We focused on the available data detailing the ECM protein profiles of human (histologically thin; hemochorial) and bovine (histologically thick; epitheliochorial) placentas and compared the shared and unique ECM proteins that are relevant to placental attachment and separation.

Nikolaos Frangogiannis: Preventing Dangerous Remodeling After Myocardial Infarction.

Nikolaos G. Frangogiannis, MD, is the Edmond J. Safra/Republic National Bank of New York Professor of Cardiovascular Medicine at the Albert Einstein College of Medicine in the Bronx. Frangogiannis graduated from the University of Athens School of Medicine in 1990 and two years later left his native Greece to pursue a residency in Internal Medicine at Baylor College of Medicine in Houston. Accepted into the Clinical Investigator Pathway, he got his first research experience in the laboratory of Mark L. Entman, MD, working with a strong group of investigators exploring myocardial inflammation in response to injury. Frangogiannis remained in Houston for 18 years, joining the faculty and establishing an independent laboratory in 1999 to expand on previous concepts on the role of inflammation in cardiac injury and repair, by probing numerous signaling pathways. The laboratory investigated whether the chemokine monocyte chemoattractant protein (MCP)-1/CCL2 was a critical regulator of myocardial infarct healing, using MCP-1 knockout mice to show that the absence of MCP-1 results in attenuated postinfarction left ventricular remodeling, but at the high cost of a prolonged inflammatory phase and delayed replacement of injured cardiomyocytes with granulation tissue.1 Using a closed-chest model of coronary infarction/reperfusion in mice, Frangogiannis’ laboratory discovered that transforming growth factor (TGF)-β/Smad3 signaling, while having antiproliferative effects, actually promotes fibrogenic actions, particularly in the infarct border zone and in the adjacent remodeling myocardium following infarction, expanding the fibrotic process into noninfarcted areas and contributing to diastolic dysfunction.2 In 2010, Frangogiannis moved to New York, where he has deployed new tools to study the cell biology of cardiac remodeling in vivo, whether following infarction or in response to other injurious stimuli, such as metabolic disease. For example, in 2013 his laboratory demonstrated that the matricellular protein thrombospondin-1 has both direct and indirect effects on the cardiac …

Role of extracellular matrix protein fibronectin‐integrin signaling in the hippocampal dentate gyrus in epilepsy

Epilepsy affects ~3 million people in the United States alone. There is no specific drug for curing epilepsy or preventing epileptogenesis, a process whereby a normal brain becomes progressively epileptic due to precipitating factors. Integrins as membrane receptors on neurons play an important role in the development of neuroplasticity in the brain. Integrin recruitment and relocalization, and extracellular matrix (ECM) protein‐integrin attachments have been reported to dynamically change their position during cell spreading and migration. We propose that ECM protein fibronectin‐integrin interaction directly affects epileptogenesis, as well as fibronectin‐integrin bond formation and cell elasticity (remodeling) are altered in epilepsy. Here, we tested this hypothesis in adult mouse hippocampal dentate granule cells (DGCs) in the kindling model of epilepsy by direct intra‐hippocampal drug infusion and immunocytochemistry, and nanoscale atomic force microscopy (AFM). The fibronectin receptor α5β1‐integrin staining intensity in DGCs from epilepsy mice was about 22% stronger than in control. Mice were treated after kindling stage 1–2 with intra‐hippocampal injection of β1‐integrin monoclonal antibody (mAb, 100 μg/ml). The normalized electrographic after discharge threshold was 2.25‐fold higher than control group. The duration of seizure behavior and the duration of electrographic afterdischarge were inhibited by 23% and 20% after β1‐integrin application, respectively (p<0.05). In AFM study, the unbinding force (25.6 ± 0.2 pN) and binding probability between fibronectin‐coated AFM probe and membrane integrins in epilepsy DGCs were 49% and 18% lower than control, respectively (p<0.05). Moreover, the cell elasticity, partially representing changes in cytoskeletal reorganization, was 66% increased in epilepsy. Treatment with α5β1‐integrin mAb significantly reversed this membrane dysfunction. These results indicate that fibronectin‐adhesive activities directly modulate development of epilepsy, and are functional dynamic and primarily bound to α5β1 integrin. Integrin signaling pathway may offer new targets for the prevention or treatment of epilepsy.Support or Funding Information(1) 2014–2015, Special Seed grant from Dr. George C.Y. Chiou and the Institute of Ocular Pharmacology, Texas A&M Health Science Center (Xin Wu ‐ PI). (2) 2013–2018, CounterACT Program, NIH, NINDS U01 NS083460 (Doodipala Reddy ‐ PI)

Stromal fibers in oral squamous cell carcinoma: A possible new prognostic indicator?

Background:Many studies have been carried out to study the role of extracellular matrix proteins, growth factors and matrix metalloproteinases on tumor invasion. However, literature related to the analysis of connective tissue fibers in varying grades of oral squamous cell carcinoma (OSCC) is very limited.Aim:To analyze the changes in collagen and elastic fibers in varying grades of (OSCC).Settings and Design:This retrospective study was carried out using a light and polarizing microscope.Materials and Methods:Three sections each were cut from fifty samples of varying grades of OSCC and ten samples of control followed by staining with H and E, Picrosirius-Red and Verhoeff–Van Gieson. Qualitative and quantitative analysis of collagen and elastic fibers were accomplished using set criteria.Statistical Analysis:Data were entered into the Statistical Package for Social Sciences (SPSS) version 13.5 for analysis.Results:A change in colors of collagen fibers was seen on progressing from well to poorly differentiated OSCC. Thin collagen fibers predominantly exhibited greenish yellow, but the thick fibers exhibited a variety of colors. As the grade of OSCC progressed, collagen fibers were loosely packed haphazardly arranged. Statistically insignificant results were obtained for quantitative analysis of collagen and qualitative analysis of elastic fibers.Conclusion:The collagen fibers undergo a change in color, orientation and packing in the stroma of varying grades of OSCC. The uniqueness of this study lies in the exploration of elastic fibers in OSCC which has not been done so far.

Abstract POSTER-BIOL-1352: Notch3 promotes anoikis resistance in ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer. Recently, a number of genome-wide studies in ovarian cancer patient samples have postulated that the course of disease can influenced by the Notch3 signaling pathway. In one study, 11% of human tumor samples showed mutation or amplification of the Notch3 receptor alone. Subsequent papers have correlated Notch3 overexpression in human samples with shorter progression-free survival as well as overall survival. In vitro studies have shown Notch3 to be involved in apoptosis resistance and chemoresistance, as well as cell cycle alterations. Together, these human studies have implicated Notch3 expression and amplification with decreased outcomes in ovarian cancer patients. To test the potential mechanism by which Notch3 over-expression promotes ovarian cancer, we examined several molecular and cellular functions of Notch3 in ovarian cancer cells. Decreasing the levels of Notch3 in ovarian cancer cells increases the susceptibility of these cells to apoptosis, which is consistent with previous studies. Using an effective siRNA-mediated knockdown of Notch3 combined with transfer to ultra-low attachment plates revealed a significant decrease in the ability of Notch3-knockdown IGROV-1 cells to remain viable in anchorage-independent conditions over 24 hours. Our data show that not only does Notch3 prevent apoptosis, but specifically promotes anoikis resistance. Further examining the mechanism underlying this novel finding revealed the positive roles of extracellular matrix proteins and FAK in promoting the anchorage-independent growth of IGROV-1 cells. Strikingly, mRNA and protein levels of col4α2 are reduced when Notch3 levels are decreased and exogenous collagen IV supplementation can largely reverse the anoikis sensitivity. Reduction of col4α2 expression by siRNA induces cell death on its own and high Notch3 expression levels correlate with higher col4α2 expression in human ovarian tumor samples. Together, these studies have revealed that Notch3 promotes anoikis resistance through col4α2 expression and may partly explain its positive correlation with poor survival in ovarian cancer patients. Citation Format: Caitlin W. Brown, Alexander S. Brodsky, Richard N. Freiman. Notch3 promotes anoikis resistance in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1352.

Clinical Drug Development Using Dynamic Biomarkers to Enable Personalized Health Care in COPD

Despite massive investments in the development of novel treatments for heterogeneous diseases such as COPD, the resources spent have only benefited a fraction of the population treated. Personalized health care to guide selection of a suitable patient population already in the clinical development of new compounds could offer a solution. This review discusses past successes and failures in drug development and biomarker research in COPD, describes research in COPD phenotypes and the required characteristics of a suitable biomarker for identifying patients at higher risk of progression, and examines the role of extracellular matrix proteins found to be upregulated in COPD. Novel biomarkers of connective tissue remodeling that may provide added value for a personalized approach by detecting subgroups of patients with active disease suitable for pharmacologic intervention are discussed.

Abstract A45: Understanding the roles of extracellular matrix proteins in pancreatic ductal adenocarcinoma progression and metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related death, and is characterized by accumulation of excessive extracellular matrix (ECM). Although the ECM of tumors, such as PDAC, has long been known to have major effects on behavior of both tumor and stromal cells, including invasion, angiogenesis, metastasis, and resistance to therapy, the detailed composition of the ECM in PDAC tumor progression and metastasis are not understood. To study the involvement of ECM proteins in PDAC primary tumor growth and the tissue-specific metastasis program, I modified the comprehensive ECM proteomic approach by Mass Spectrometry (MS) recently developed in our lab to profile the ECM changes during PDAC progression in both the LSL-KrasG12D/+; LSL-Trp53R127H/+; Pdx-1-Cre (KPC) mouse models of PDAC and human patient samples. We will compare ECM profiles from normal pancreas, pancreatitis, early and late pancreatic intraepithelial neoplasia (PanIN) stages, and PDAC. Initial MS identified between 100 and 140 ECM proteins in samples representing each stage. The combination of mouse models and human samples will allow me to uncover critical ECM changes during PDAC progression and metastasis and, furthermore, to investigate and understand the functional consequences of such changes. The ongoing studies will characterize the ECM of PDAC in detail and provide novel insights into diagnosis, prognosis and treatments of this very difficult disease. Citation Format: Chenxi Tian, Daniel Olund, David Tuveson, Richard O. Hynes. Understanding the roles of extracellular matrix proteins in pancreatic ductal adenocarcinoma progression and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A45.

Transdifferentiation of Bone Marrow Mesenchymal Stem Cells into the Islet-Like Cells: the Role of Extracellular Matrix Proteins.

Pancreatic islet implantation has been recently shown to be an efficient method of treatment for type 1 diabetes. However, limited availability of donor islets reduces its use. Bone morrow would provide potentially unlimited source of stem cells for generation of insulin-producing cells. This study was performed to evaluate the influence of extracellular matrix proteins like collagen, laminin, and vitronectin on bone marrow mesenchymal stem cells (BM-MSCs) transdifferentiation into islet-like cells (ILCs) in vitro. To our knowledge, this is the first report evaluating the importance of vitronectin in transdifferentiation of BM-MSCs into ILCs. Rat BM-MSCs were induced to ILCs using four-step protocol on plates coated with collagen type IV, laminin type I and vitronectin type I. Quantitative real-time PCR was performed to detect gene expression related to pancreatic β cell development. The induced cells expressed islet-related genes including: neurogenin 3, neurogenic differentiation 1, paired box 4, NK homeobox factor 6.1, glucagon, insulin 1 and insulin 2. Laminin but not collagen type IV or vitronectin enhanced expression of insulin and promoted formation of islet-like structures in monolayer culture. Laminin triggered transdifferentiation of BM-MSCs into ILCs.

Role of extracellular matrix proteins, matrix metalloproteinase activity and arterial stiffness in ascending aorta dilatation

Objectives: The purpose of this study was to evaluate potential mechanisms promoting ascending aorta aneurysm (AAA) development in the pts with different pathology. Design and methods: Aortic tissue samples were collected during operation from 50 pts with AAA: 16 pts with atherosclerosis (AS) (mean age 60.1±1.6 yrs; 1.5:1, ratio m:f), 22 pts with bicuspid aortic valves (BAV) (mean age 55.7±1.9 yrs; 1.6:1, ratio m:f) and 12 pts with tricuspid valves (TAV) (mean age 50.4±4.3 yrs; 1.7:1, ratio m:f). Tissue samples were collected from 17 pts without aortic pathology (mean age 56.5±3.5 yrs). Matrix metalloproteinase (MMP) -2,-9 activities were assessed in aortic tissue homogenates by substrate-specific zymographic analysis. The relative abundances of latent MMP-2,9, collagen I, elastin and fibrillin were determined by quantitative immunoblotting techniques. Arterial stiffness was assessed by SphygmoCor device (Australia) using indirect carotid-femoral distance. The normal value of pulse wave velocity (PWV) was considered below 10 m/s. Results: There were no significant differences in thoracic aorta diameter between the groups (65.4±13.3 mm in AS, vs 58.0±4.8 mm in BAV vs 64.1±12.9 mm in TAV). Latent MMP2 was increased in all AAA groups compared with controls (BAV, TAV and AS p=0.003, p=0.029, p=0.007, respectively). But latent MMP9 was higher only in BAV group (p=0.04). Aortic dilatation in pts with BAV was associated with increased active MMP-9 level (r=0.66, p=0.037). There was increased the collagen/elastin ratio in all pts with AAA, but significant only in BAV (3.2±2.3) compared with controls (1.1±0.5, p=0.018). An increase of collagen content was correlated with active MMP9 level in pts with AS (r=0.559, p=0.037), whereas change of collagen/elastin ratio in pts with BAV was associated with increased latent MMP2 (r=0.608, p=0.02). Tendency to increase of latent and active MMP9 level was related with increase of index augmentation in all groups. Conclusions: Composition of the extracellular matrix determines the MMP activity and plays an important role in the pathogenesis of AAA. More significant changes in the composition of the extracellular matrix proteins may explain the rapid increase in the aorta diameter in pts with BAV. Arterial stiffness can be used as a marker for noninvasive monitoring of patients with aortic dilatation.

Report on the Myomatrix Conference April 22–24, 2012, University of Nevada, Reno, Nevada, USA

The Myomatrix 2012 conference held April 22-24th, 2012 at the University of Nevada, Reno convened 73 international participants to discuss the dynamic relationship between muscle and its matrix in muscular dystrophy with a specific focus on congenital muscular dystrophy. Seven sessions over 2½ days defined three central themes: (1) the role of extracellular matrix proteins and compartments in development and specifically in congenital muscular dystrophy (CMD) (2) the role of extracellular matrix signaling and adhesion to membrane receptors and (3) the balance and interplay between inflammation and fibrosis as drivers of altered matrix stiffness, impaired regeneration and progressive dystrophy. This report highlights major conference findings and the translational roadmap as defined by conference attendees.

Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction

The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission.

Quantitative Glycoproteomic Analysis of Optimal Cutting Temperature-Embedded Frozen Tissues Identifying Glycoproteins Associated with Aggressive Prostate Cancer

Prostate cancer is the most common malignancy in men in the United States, and one in seven men with prostate cancer dies of the disease. A major issue of prostate diagnosis is that there is no good method to reliably distinguish aggressive prostate cancer from nonaggressive prostate cancer. This leads to significant unnecessary suffering among prostate cancer patients and massive unnecessary health care expenditures. In this study, we aim to identify glycoproteins associated with aggressive prostate cancer using optimal cutting temperature (OCT)-embedded frozen tissues obtained from patients with known clinical outcome. To eliminate the interference of mass spectrometric analysis by the compounds in OCT and identify extracellular proteins that are likely to serve as biomarkers in body fluids, we employed glycoproteomic analysis using solid-phase extraction of glycopeptides, which allowed the immobilization of glycopeptides to solid support and removal of OCT from sample proteins before releasing the glycopeptides from the solid support for mass spectrometry analysis. Tumor tissues were cryostat microdissected from four cases of aggressive and four cases of nonaggressive prostate tumors, and glycopeptides were isolated and labeled with iTRAQ reagents before the samples were analyzed with LTQ Orbitrap Velos. From the aggressive prostate cancer tissues, we identified the overexpression of three glycoproteins involved in an extracellular matrix remodeling and further examined two glycoproteins, cathepsin L and periostin, using Western blot and immunohistochemistry analyses. This is the first proteomic study to identify proteins potentially associated with aggressive prostate cancer using OCT-embedded frozen tissues. Further study of these proteins will be needed to understand the roles of extracellular matrix proteins in cancer progression and their potential clinical utility in improving diagnosis of aggressive prostate cancer.

The extracellular matrix of the dermis: flexible structures with dynamic functions

The current understanding of the role of extracellular matrix proteins is mainly based on their structural properties and their assembly into complex networks. The multiplicity of interactions between cells, cytokines and growth factors within the networks determines functional units dictating the biophysical properties of tissues. This review focuses on the understanding how alterations in the genes, modifying enzymes or biological functions of extracellular matrix molecules, lead to inborn or acquired skin disorders. Analysis of the disease mechanisms provides the basis for the emerging concept that not solely structural defects of single extracellular matrix proteins are at fault, but rather that the functional unit as a whole is not working properly, causing similar clinical symptoms although the causative genes are entirely different. The understanding of these disease-causing pathways has already led to surprising new therapeutic developments applied to rare inborn disorders. They now permit to design new concepts for the treatment of more common diseases associated with the accumulation of connective tissue and alterations of the biomechanical properties of the extracellular matrix.

The role of ECM proteins and protein fragments in guiding cell behavior in regenerative medicine

The promise of biomaterials design for regenerative medicine tissue engineering is predicated on the fundamental ability to direct or guide specific and highly coordinated cellular behaviors that culminate in the creation of physiologically functional tissues and organs. To date, our efforts have focused primarily on the grafting and presentation of short synthetic peptides with just cause. Short peptides are capable of high levels of control, can be manufactured relatively easily in a highly reproducible manner under GMP guidelines and are readily modified to enable their integration with numerous current and emerging chemistries for biomaterials grafting. However, while extracellular matrix (ECM)-derived peptides have demonstrated their initial purpose of promoting cell adhesion, their general lack of specificity and significantly decreased receptor binding affinities have proven detrimental in attempts to regulate highly specific and integrated processes necessary for tissue regeneration. Unlike adhesion peptides, the natural ECM displays a complex interplay with cells by supporting environmentally sensitive and cell dependent integrin specificity and binding affinity. Furthermore, the adhesion ligands on ECM proteins display a finely tuned and evolutionarily directed spatial periodicity, of which is dynamically controlled through both mechanical and chemical modifications. These and other emerging concepts from matrix biology require our attention if biomaterials design is to fulfill its promise. Here, we are charged with debating the statement ‘The use of short synthetic adhesion peptides, like RGD, is the best approach in the design of biomaterials that guide cell behavior for regenerative medicine tissue engineering’. In this Leading Opinion Paper I will focus on aspects of natural ECM proteins and protein fragments that have proven difficult, if not impossible to date, to recapitulate in peptide-based systems. While this represents an argument against the use of peptides per se, it might also be viewed as outlining the challenges and opportunities for the biomaterials field.

Characterization of drCol 15a1b: A Novel Component of the Stem Cell Niche in the Zebrafish Retina

There is a clear need to develop novel tools to help improve our understanding of stem cell biology, and potentially also the utility of stem cells in regenerative medicine. We report the cloning, functional, and bioinformatic characterization of a novel stem cell marker in the zebrafish retina, drCol 15a1b. The expression pattern of drCol 15a1b is restricted to stem cell niches located in the central nervous system, whereas other collagen XVs are associated with muscle and endothelial tissues. Knocking down drCol 15a1b expression causes smaller eyes, ear defects, and brain edema. Microscopic analysis reveals enhanced proliferation in the morphant eye, with many mitotic nuclei located in the central retina, together with a delayed differentiation of the mature retinal cell types. Besides, several markers known to be expressed in the ciliary marginal zone display broader expression areas in morpholino-injected embryos, suggesting an anomalous diffusion of signaling effectors from the sonic hedgehog and notch pathways. These results indicate that drCol 15a1b is a novel stem cell marker in the central nervous system that has a key role in homing stem cells into specialized niches in the adult organism. Moreover, mutations in the hCol 18a1 gene are responsible for the Knobloch syndrome, which affects brain and retinal structures, suggesting that drCol 15a1b may function similarly to mammalian Col 18a1. Thus, our results shed new light on the signaling pathways that underlie the maintenance of stem cells in the adult organism while helping us to understand the role of extracellular matrix proteins in modulating the signals that determine stem cell differentiation, cell cycle exit and apoptosis.

Abstract 1437: Laminin-1 confers Bleomycin-resistance through poly(ADP-ribose) polymerase activation in 3D culture

Abstract The purpose of this study is to examine the role of extracellular matrix proteins in regulating Bleomycin resistance and to evaluate the potential role of poly(ADP-ribose) polymerase (PARP) in modulating drug resistance in MDA-MB-231 breast cancer cells. These phenomenons were investigated using a 3D culture model where the MDA-MB-231 cells were cultured on either collagen I or laminin-1. Cells were evaluated for viability as a dose-response to the chemotherapeutic agent, Bleomycin. Cell lysates were also evaluated for PARP expression as a consequence of the 3D culture environment. Cells were then treated with the PARP inhibitor, PJ-34, and evaluated for viability as a dose-response to Bleomycin in the 3D cultures. PARP activity was assessed using a pharmacodynamic ELISA. For the first time, this study presents evidence that the basement membrane protein, laminin-1, induces Bleomycin resistance, that laminini-1 induces PARP expression and activity, and that PARP inhibition sensitizes breast cancer cells to Bleomycin treatment. In conclusion, this data provides a mechanism for ECM-mediated Bleomycin-resistance and the potential therapeutic application for PARP inhibitors to sensitize tumors to Bleomycin treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1437.

The Integrin-blocking Peptide RGDS Inhibits Airway Smooth Muscle Remodeling in a Guinea Pig Model of Allergic Asthma

Airway remodeling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. The mechanisms driving these changes are, however, incompletely understood. Recently, an important role for extracellular matrix proteins in regulating ASM proliferation and contractility has been found, suggesting that matrix proteins and their integrins actively modulate airway remodeling. To investigate the role of RGD (Arg-Gly-Asp)-binding integrins in airway remodeling in an animal model of allergic asthma. Using a guinea pig model of allergic asthma, the effects of topical application of the integrin-blocking peptide RGDS (Arg-Gly-Asp-Ser) and its negative control GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) were assessed on markers of ASM remodeling, fibrosis, and inflammation induced by repeated allergen challenge. In addition, effects of these peptides on human ASM proliferation and maturation were investigated in vitro. RGDS attenuated allergen-induced ASM hyperplasia and hypercontractility as well as increased pulmonary expression of smooth muscle myosin heavy chain and the proliferative marker proliferating cell nuclear antigen (PCNA). No effects were observed for GRADSP. The RGDS effects were ASM selective, as allergen-induced eosinophil and neutrophil infiltration as well as fibrosis were unaffected. In cultured human ASM cells, we demonstrated that proliferation induced by collagen I, fibronectin, serum, and platelet-derived growth factor requires signaling via RGD-binding integrins, particularly of the alpha(5)beta(1) subtype. In addition, RGDS inhibited smooth muscle alpha-actin accumulation in serum-deprived ASM cells. This is the first study indicating that integrins modulate ASM remodeling in an animal model of allergic asthma, which can be inhibited by a small peptide containing the RGD motif.

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.

The roles of extracellular matrix proteins, apoptosis and c-kit positive cells in the pathogenesis of ureteropelvic junction obstruction

To investigate histopathological changes in ureteropelvic junction obstruction (UPJO) from an etiological perspective. Medical records of patients with UPJO were reviewed and pathological specimens collected. Nephrectomy materials from forensic autopsies were taken as controls. Specimens were assessed with light microscopy. Fibronectin, type 4 collagen, laminin, Bax and Bcl-2 expression for apoptosis, together with interstitial cells of Cajal determination with c-kit were determined immunohistochemically. Staining scores were evaluated semiquantitatively. Results were evaluated using Mann-Whitney U-test. Control group comprised 14 children (median age, 3.5 years; 6 months-17 years). Study group comprised 22 children with UPJO (median age, 9 months; 1 month-10 years). Light microscopy revealed non-specific inflammation, epithelial proliferation and atrophy with fibrosis in the smooth muscle of the UPJ in all patients. Fibronectin, type 4 collagen and laminin were found to be significantly increased in UPJO at the intrafascicular space of smooth muscle and the matrix of stroma. Bcl-2 expression was increased in UPJO. c-Kit was unable to stain interstitial cells of Cajal, but staining for mast cells was significant. High expression of fibronectin, laminin and type 4 collagen may indicate a relation to the pathogenesis of UPJO. Defective kidney morphogenesis, during branching and tubulogenesis of ureteric bud, may be responsible for this congenital pathology.