Localization Of Extracellular Matrix Proteins Research Articles

Abstract 1269: Evaluating the effect of metformin on components of extracellular matrix in colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide, and is associated with high morbidity and mortality. Approximately 50% of all CRC patients develop metastases. Tumor microenvironment (TME), including extracellular matrix (ECM), plays an important role in tumor progression and metastasis in CRC, indicating an important role of agents targeting ECM in the treatment of CRC. ECM acts as niche to support cancer cells. In this study, the role of metformin as a therapeutic agent in modulating ECM components of CRC has been studied. Methods: HCT116 (aggressive, poorly differentiated) and HT 29 (less aggressive, moderately differentiated), human CRC cell lines were treated with different doses of metformin (0.1, 2.5 and 5 mM) for 14 days in vitro and subsequently microarray analysis was done to identify differentially expressed genes. To observe the role of metformin in modulating the ECM, ECM-receptor interaction pathway was given primary focus among the list of pathways identified by DAVID (Database for Annotation, Visualization and Integrated Discovery) bioinformatics resources. The seven differentially expressed genes of ECM-receptor interaction pathway (elucidated by microarray) were -COL3A1, COL4A2, COL5A2, VTN, ITGA6, TNXB and LAMC3. These genes were validated using RT-qPCR in both the cell lines. Protein expression was analyzed by Western blotting. Immunohistochemistry (IHC) was done in different histopathological grades of CRC samples to evaluate the expression and localization of ECM proteins. Results: The gene expression of COL4A2, COL5A2, ITGA6, VTN and TNXB by RT-qPCR was decreased with metformin treatment and these results were consistent with microarray data. However, discrepancy was observed in the gene expression of COL3A1 and LAMC3 between microarray and RT-qPCR. Expression of COL3A1, VTN was found to be increased whereas a decrease in expression of ITGA6 was observed in different histopathological grades of CRC samples with IHC. The expression of PI3K and AKT proteins were decreased in HCT116 cells with metformin treatment justifying its anticancer properties. Conclusions: Anticancer role of metformin via mTOR and PI3K-AKT signaling has been studied before, but this study identifies the novel ECM proteins in CRC that can be modulated by metformin. The results from this study indicates the promising role of metformin in CRC as it is associated with very less side effects and can influence various aspects of cancer biology. Citation Format: Amar Preet Kaur, Aditi Bhattacharya, Muzaffer Ahmed Bhat, Aakriti Bansal, Sudip Sen. Evaluating the effect of metformin on components of extracellular matrix in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1269.

Absence of collagen IX accelerates hypertrophic differentiation in the embryonic mouse spine through a disturbance of the Ihh-PTHrP feedback loop

Collagen IX (Col IX) is a component of the cartilage extracellular matrix and contributes to its structural integrity. Polymorphisms in the genes encoding the Col IX ɑ2- and ɑ3-chains are associated with early onset of disc degeneration. Col IX-deficient mice already display changes in the spine at the newborn stage and premature disc degeneration starting at 6months of age. To determine the role of Col IX in early spine development and to identify molecular mechanisms underlying disc degeneration, the embryonic development of the spine was analyzed in Col IX -/- mice. Histological staining was used to show tissue morphology at different time points. Localization of extracellular matrix proteins as well as components of signaling pathways were analyzed by immunohistochemistry. Developing vertebral bodies of Col IX -/- mice were smaller and already appeared more compact at E12.5. At E15.5, vertebral bodies of Col IX -/- mice revealed an increased number of hypertrophic chondrocytes as well as enhanced staining for the terminal differentiation markers alkaline phosphatase and collagen X. This correlates with an imbalance in the Ihh-PTHrP signaling pathway at this time point, reflected by an increase of Ihh and a concomitant decrease of PTHrP expression. An accelerated hypertrophic differentiation caused by a disturbed Ihh-PTHrP signaling pathway may lead to a higher bone mineral density in the vertebral bodies of newborn Col IX -/- mice and, as a result, to the early onset of disc degeneration.

Effect of a nutrient mixture on the localization of extracellular matrix proteins in HeLa human cervical cancer xenografts in female nude mice

Cervical cancer is one of the most commonly diagnosed cancers and a significant cause of mortality in women worldwide. Although cervical cancer is fully treatable in the early stages, once it has metastasized, patient outcome is poor. The objective of the present study was to investigate the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on the expression of extracellular matrix (ECM) proteins in HeLa cell xenografts in nude female mice. After housing for 1 week, female athymic nude mice between 5 and 6 weeks of age (n=12) were inoculated subcutaneously with 3×106 HeLa cells in phosphate-buffered saline and Matrigel and randomly divided into two groups. These were the control group, in which the mice were fed with regular mouse chow, and the NM group, in which the mice were fed with the regular diet supplemented with 0.5% NM (w/w). After 4 weeks, the tumors were excised and processed for histology. Tumor growth was evaluated and the tumors were stained for the ECM proteins collagen I, collagen IV, fibronectin, laminin, periodic acid-Schiff (PAS) and elastin. NM strongly inhibited (by 59%, P=0.001) the growth of HeLa xenografts in nude mice. Tumors from control mice exhibited little to no collagen I expression either internally or in the fibrous capsule, while tumors from the NM group expressed collagen I in the fibrous capsule and within the tumor. Tumors from the control group showed diffuse cytoplasmic and capsular collagen IV with abundant nucleated cells. NM treatment substantially increased collagen IV production and induced a dense fibrous network of collagen IV with chambers that surrounded live nucleated cells and large amounts of necrotic cell debris. Tumors from the mice fed with the NM exhibited a well-defined border of fibronectin in the capsule and intense areas of staining internally whereas control group tumors showed less overall fibronectin with sporadic internal staining and little in the fibrous capsule. Although laminin appeared abundantly in control and NM-treated tumors, the NM group tumors exhibited a chamber-like network of laminin internally. Tumors from the control group exhibited internal areas of intense PAS staining, whereas tumors from the NM-treated group exhibited a more uniform diffuse pattern of PAS staining. In conclusion, NM supplementation of HeLa xenograft-bearing female nude mice demonstrated a potent inhibition of tumor growth and enhancement of ECM proteins, suggesting the therapeutic value of this specific nutrient complex in the treatment of cervical cancer.

Ultrastructural localization of extracellular matrix proteins of the lymph node cortex: evidence supporting the reticular network as a pathway for lymphocyte migration

BackgroundThe lymph node (LN) is a crossroads of blood and lymphatic vessels allowing circulating lymphocytes to efficiently recognize foreign molecules displayed on antigen presenting cells. Increasing evidence indicates that after crossing high endothelial venules, lymphocytes migrate within the node along the reticular network (RN), a scaffold of fibers enwrapped by fibroblastic reticular cells (FRC). Light microscopy has shown that the RN contains specific extracellular matrix (ECM) proteins, which are putative molecular "footholds" for migration, and are known ligands for lymphocyte integrin adhesion receptors.ResultsTo investigate whether ECM proteins of the RN are present on the outer surface of the FRC and are thus accessible to migrating lymphocytes, ultrastructural immunohistochemical staining of cynomolgus monkey LN was performed using antibodies to human ECM proteins that were successfully employed at the light microscopic level. The fibrillar collagens I and III were observed primarily within the reticular network fibers themselves. In contrast, the matrix proteins laminin, fibronectin, collagen IV, and tenascin were observed within the reticular fibers and also on the outer membrane surface of the FRC.ConclusionsThese findings suggest a molecular basis for how the RN functions as a pathway for lymphocyte migration within the lymph node.

Mixing model systems: Using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development

Human vestibular dysfunction is an increasing clinical problem. Degeneration or displacement of otoconia is a significant etiology of age-related balance disorders and Benign Positional Vertigo (BPV). In addition, commonly used antibiotics, such as aminoglycoside antibiotics, can lead to disruption of otoconial structure and function. Despite such clinical significance, relatively little information has been compiled about the development and maintenance of otoconia in humans. Recent studies in model organisms and other mammalian organ systems have revealed some of the proteins and processes required for the normal biomineralization of otoconia and otoliths in the inner ear of vertebrates. Orchestration of extracellular biomineralization requires bringing together ionic and proteinaceous components in time and space. Coordination of these events requires the normal formation of the otocyst and sensory maculae, specific secretion and localization of extracellular matrix proteins, as well as tight regulation of the endolymph ionic environment. Disruption of any of these processes can lead to the formation of abnormally shaped, or ectopic, otoconia, or otoconial agenesis. We propose that normal generation of otoconia requires a complex temporal and spatial control of developmental and biochemical events. In this review, we suggest a new hypothetical model for normal otoconial and otolith formation based on matrix vesicle mineralization in bone which we believe to be supported by information from existing mutants, morphants, and biochemical studies.

Changes in epithelial cell turnover and extracellular matrix in human small intestine after TPN

Background. The atrophy and architectural remodeling of the jejunal mucosa arising in adults receiving total parenteral nutrition (TPN) has been suggested to originate from a disturbance in tissue homeostasis. The present study aims at examining (1) whether there are differences in proliferation and apoptosis of epithelial cells between enterally and parenterally nourished patients and (2) whether the distribution pattern of extracellular matrix (ECM) proteins known to influence cell turnover along the the crypt-villus axis is changed after TPN.Methods. The mitotic frequency and the proliferation index [using an antibody against Ki-67 antigen (MIB 1)] were determined on epoxy semithin and paraffin sections, respectively. Morphological techniques and the TUNEL assay were applied to detect apoptotic events. Immunolocalization of collagen IV, laminin, fibronectin, tenascin, and collagen VI was performed on cryosections.Results. After TPN the cell renewal was significantly enhanced, while epithelial cell death was drastically reduced. The comparison of TPN and EN patients revealed differences in the distribution patterns of the ECM proteins laminin, fibronectin, and tenascin along the crypt-villus axis. Moreover, after TPN an increased expression of collagen types IV and VI was observed.Conclusions. TPN in human adults is associated with alterations in epithelial cell turnover and changes in expression and/or localization of ECM proteins. Thus, the inverted route of nutrient supply in patients might modify environmental tissue conditions, which may influence the interactions between intestinal epithelial cells and the extracellular matrix.

Composition of the extracellular matrix of the peritoneum.

To localize the extracellular matrix proteins collagen I, collagen IV, fibronectin, and laminin in the peritoneal membrane. Peritoneal biopsies (n = 13) from the anterior abdominal wall and the uterine serosa (n = 3) were incubated with antibodies to collagen IV, laminin, collagen I, and fibronectin. Specimens were examined using light and confocal laser scanning microscopy. All of the extracellular matrix (ECM) proteins were present immediately under the mesothelium. Collagen (Col) IV and laminin (LM) were seen in the smooth muscle of microvascular structures, in the subendothelial basement membrane, and were present in a fascicular pattern in the peritoneal stroma. Collagen I was distributed diffusely in the peritoneal stroma. Fibronectin was also present in the subendothelial basement membrane. The resolution of the confocal microscope allowed for localization of extracellular matrix proteins in relation to the mesothelium. The presence of collagen IV, laminin, collagen I, and fibronectin under the mesothelium suggests that cells invading the peritoneum must have the ability to degrade and remodel this matrix.

Confocal analysis of the dystrophin protein complex in muscular dystrophy.

The dystrophin protein complex (DPC), composed of at least 10 proteins that associate with dystrophin, is critical for the maintenance of normal muscle fiber structure and physiology. In this study, we used immunohistochemistry and confocal microscopy to examine the relative abundance and distribution of several of these proteins in muscle biopsies taken from patients with various muscular dystrophies. The optical sectioning capability of confocal microscopy allowed us to comprehensively analyze the semiquantitative expression of components of the DPC. Alpha-sarcoglycan-deficient patients displayed a marked reduction in membrane immunostaining of the sarcoglycan complex. Gamma-sarcoglycan-deficient patients showed variable decreased immunostaining of the sarcoglycan complex proteins. When beta-sarcoglycan was expressed appropriately at the sarcolemma of gamma-sarcoglycan-deficient patients, intracellular labeling of beta-sarcoglycan was also present. Beta-sarcoglycan-deficient patients showed poor localization of extracellular matrix proteins in addition to a complete absence of the sarcoglycans. Merosin-deficient patients showed relatively normal immunostaining levels of all other members of the DPC. Finally, dystrophin-deficient patients showed little or no change in the expression of extracellular matrix proteins; however, some sarcoglycans were significantly decreased. These data allowed us to suggest unique fundamental interactions between the members of the DPC.

Comparison of Stromal Remodeling and Keratocyte Response After Corneal Incision and Photorefractive Keratectomy

Purpose: We investigated the keratocyte response and stromal remodeling after corneal incision and photorefractive keratectomy to understand the histophysiological and immunohistochemical differences between these two types of surgery. Methods: Corneal incision or photorefractive keratectomy was performed in rabbits or rats. Then we chronologically observed the histological changes and the changes in the localization of extracellular matrix proteins. Results: In both types of surgery, the keratocyte population at the damaged stroma became sparse, and the cells began undergoing apoptosis immediately after the surgical procedure. Subsequently, activated keratocytes migrated into the acellular zone, and the cells formed multiple layers at the resurfaced subepithelial regions. We observed deposition of amorphous substances between keratocytes that had migrated, and stromal remodeling appeared to start. Three months after surgery, the corneal structure had recovered to a near-normal condition at the corneal incision. After photorefractive keratectomy, however, extracellular matrix proteins were strongly immunoreactive at the subepithelial regions. Conclusions: These results suggest that the stromal wound-healing processes are similar after corneal incision and photorefractive keratectomy. A corneal incision may induce a transient keratocyte response during stromal remodeling, but photorefractive keratectomy may induce a sustained keratocyte response.

Localization of Extracellular Matrix Proteins after Holmium YAG Laser Radiation in Rat Cornea

Purpose: To understand corneal responses to holmium YAG (Ho:YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. Methods: Rats were radiated with an Ho:YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin. Results: One day after Ho:YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. Conclusions: These results suggest that Ho:YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.

Immunohistochemical localization of extracellular matrix proteins in luteal phase endometrium of fertile and infertile patients.

The lack of expression of certain components involved in cell adhesion and migration is believed to contribute to endometrial dysfunction and implantation failure. The purpose of this study was to investigate whether luteal phase endometrium in women with unexplained infertility differs, with respect to specific extracellular matrix (ECM) proteins, from endometrium of normal fertile women. A panel of monoclonal antibodies to collagen type IV, fibronectin and laminin was used to characterize the localization of ECM components in the different endometrial compartments. Precisely timed endometrial biopsies obtained at 4, 7, 10 and 13 days following the luteinizing hormone surge were obtained from 22 normal fertile women (group 1) and 24 women suffering from unexplained infertility (group 2). Paraffin-embedded sections were labelled using the streptavidin-biotin alkaline phosphatase technique. In group 1, collagen type IV, fibronectin and laminin were absent from the luminal epithelium but present in stromal cells and the basement membrane of glands and blood vessels. In group 2, these components were absent from all endometrial regions using equivalent titres of antibody to those used in group 1. This suggests that the endometrium of women with unexplained infertility demonstrates defects in the distribution of certain ECM glycoproteins. A possible consequence of this defect may be implantation failure.

Immunohistochemical localization of extracellular matrix proteins in human glioma, both in vivo and in vitro.

Expression of type IV collagen, fibronectin and laminin in various types of primary human brain tumor sections and normal brain tissue sections as well as cultured glioma cell lines was examined by an immunofluorescence technique. Type IV collagen, fibronectin, and laminin were mainly localized to the basement membrane of the vasculature in glioblastoma, anaplastic astrocytoma, low grade glioma, and in normal brain. However, positive staining for all the extracellular matrix (ECM) components tested was found only in glioblastoma sections both in the cells and in the ECM. In all other tumor types and in normal brain tissue, the cells did not stain for any of the ECM components. Four glioblastoma cell lines and autologous ECM synthesized by respective glioblastoma cell lines also showed positive staining for type IV collagen, fibronectin and laminin in vitro. These results suggest that glioblastoma cells both in vitro and in vivo express the extracellular matrix components that are involved in the regulation of tumor cell invasion.

Immunohistochemical localization of extracellular matrix proteins in cerebral vessels in chronic hypertension.

Vascular hypertrophy or vascular remodeling are both associated with an increase in the extracellular matrix of cerebral arteries and arterioles in chronic hypertension. Rats with chronic renal hypertension were studied to determine whether the extracellular matrix proteins--collagen IV, laminin, and fibronectin--are increased in the thick-walled cerebral vessels in brain and those associated with areas of blood-brain barrier (BBB) breakdown to protein. The latter was detected by extravasation of endogenous serum proteins in brain. Serum proteins and the extracellular matrix proteins--fibronectin, laminin, and collagen IV--were detected by immunohistochemistry. Hypertensive rats having blood pressures over 150 mm Hg showed mild to moderate degrees of mural thickening of pial and intracerebral arterioles. In addition, these vessels demonstrated increased immunoreactivity with collagen IV, fibronectin, and laminin antisera. This occurred concomitant with the development of hypertension and prior to the observation of BBB breakdown to protein. Four rats having mean maximum systolic blood pressures in excess of 220 mm Hg developed multifocal areas of increased vascular permeability to endogenous serum proteins in the boundary zones of the territories supplied by the major cerebral arteries. The arterioles in these areas showed a severe degree of vascular thickening, which was due to medial hyperplasia/hypertrophy and increased mural deposition of serum proteins and the extracellular matrix proteins--laminin, fibronectin, and collagen IV. This study demonstrates that extracellular matrix proteins play an important role in the vascular response to increased intraluminal pressure. However, since this change occurs in diseases in the absence of hypertension it should be regarded as a nonspecific response of cellular components of vessel walls to injury.

Altered expression of extracellular matrix proteins and integrins in oral lichen planus (OLP).

The immunohistochemical distribution of collagens type I, III, IV, V, VI, of undulin and tenascin, and of integrins alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 4, was studied in 14 biopsies of oral lichen planus (OLP), 5 biopsies of orthokeratinized gingiva and 4 biopsies of oral fibrous hyperplasia. The localization of extracellular matrix proteins showed altered expression in OLP when compared to normal oral mucosa, with two principal patterns corresponding to the reticular or atrophic type. Whereas in the reticular type a focal loss of immunoreactivity for collagen types I, III, V, VI and undulin was noted in areas with a cellular infiltrate, in the atrophic variant almost complete loss of immunoreactivity of the subepithelial extracellular matrix was found. There was no clear correlation between the distribution of extracellular matrix proteins and their integrin receptors. The present findings suggest that the autoimmune reaction in OLP might not be primarily targeted to oral keratinocytes but to an unknown antigen in the connective tissue stroma. The changes in the subepithelial extracellular matrix associated with the inflammatory reaction might, especially in the atrophic variant, impair the cross-talk between epithelium and mesenchyme and favour both the loss of barrier function and the development of erosions in the clinical course of the disease.

Modulation of extracellular matrix proteins in rat liver during development

The expression and localization of extracellular matrix proteins in rat liver was investigated as a function of liver development. Levels of extracellular matrix proteins were measured by dot-blot or immunoblot protocols using monospecific antibodies prepared against collagen types I, III and IV; laminin; fibronectin; and fibronectin receptor. Proline and hydroxyproline levels from extracted liver collagen were quantitated by Pico Tag analysis. It was observed that the content of type IV collagen and fibronectin in the rat liver increased two to four times during the perinatal period. In contrast, levels of laminin and collagen types I and III decreased up to twofold in developing rat livers. The content of fibronectin receptor during ontogeny was decreased four times in an inverse relationship to fibronectin molecules. Fibronectin receptor and extracellular matrix proteins displayed no difference in apparent molecular weight as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots. Indirect immunofluorescence staining of frozen thin liver sections revealed that the pattern of localization of extracellular matrix proteins in the nonvascular regions of fetal liver was punctate rather than restricted to a specific region such as the perisinusoidal area of adult livers. Similarly, fibronectin receptor was also present, mainly in the sinusoidal area of adult livers, whereas fetal sections were diffusely stained. Our findings suggest that the differential modulation of extracellular matrix proteins and their localization in the developing rat livers undergo a dramatic alteration in the composition and structural organization of matrix material, which may act to modulate proliferation and to promote the differentiation of liver cells during development.

Ultrastructural localization of extracellular matrix proteins in liver biopsies using ultracryomicrotomy and immuno‐gold labelling

We describe a technique for the localization of extracellular matrix proteins in wedge and needle biopsy specimens of human liver. Using ultra-thin (50-70 nm) sections of puncture perfusion fixed tissue, extracellular matrix proteins were localized using a protein A-gold labelling procedure. We obtained good preservation of ultrastructural detail and specific labelling for collagen types I, III and IV and fibronectin. The method represents a sensitive means of identifying the extracellular distribution and the cellular origin of these proteins in normal and diseased human liver.