Prominent Extracellular Matrix Research Articles

Investigating the correlation between the protein adhesion simulation and the biocompatibility of polymeric substrate for skin-tissue-engineering applications.

Investigating the protein adhesion properties of polymeric scaffolds through computational simulations can predict the biocompatibility of scaffolds before an experimental assay is carried out. This prediction can be highly beneficial since it can cut costs and the time it takes for experimental assays. The current study aims to test the hypothesis that there is a correlation between the biocompatibility of a composite scaffold and the molecular dynamics simulations of protein adhesion. To this end, chitosan and gelatin were selected for fabricating a composite skin-tissue wound scaffold with five different polymer ratios. This polymeric blend has not been simulated for protein adhesion. The cell proliferation and viability of the samples were quantified via MTT assay using fibroblast cells. Then a series of molecular dynamics simulations were performed to measure the adhesion energy of two prominent extracellular matrix proteins - fibronectin, and collagen type I. Besides, a higher gelatin percentage in the scaffold leads to a decrease in the porosity. The results demonstrated a strong correlation between the experimental data and molecular dynamics simulations. The sample with equal amounts of chitosan and gelatin had the highest cell viability and the strongest adhesion energy, of 239 kcal mol-1 for collagen type I, and 149 kcal mol-1 for fibronectin. This correlation was also evident in other samples: samples with gelatin-to-chitosan ratios of 3 : 1 and 1 : 3 had the lowest cell viability and the weakest adhesion energy, respectively.

Correlation between Fibrin Fibrillation Kinetics and the Resulting Fibrin Network Microstructure.

Fibrin, the prominent extracellular matrix in early wound tissue, is discussed to influence immune cells and healing. The nature of fibrinogen/fibrin to form fibrillary networks is frequently exploited to engineer microenvironments for cellular analysis. This study focuses on revealing the correlation of fibril formation kinetic and the resulting network microstructure of engineered 3D fibrin networks. Different concentrations of fibrinogen (1-3mgmL-1 ), thrombin (0.01-0.15UmL-1 ), sodium chloride (40-120mm), and calcium chloride (1-10mm) are applied to assess the impact on the fibril growth kinetics by turbidity analysis and on the resulting fibril and pore diameter by laser scanning microscopy. The results highlight a direct influence of the sodium chloride concentration on fibrillation kinetics and reveal a strong correlation between fibrillation kinetics and network microstructure. With the assumption of a first-order growth kinetic, an increase of the growth constant k (0.015-0.04min-1 ) is found to correlate to a decrease in fibril diameter (1-0.65µm) and pore diameter (11-5µm). The new findings enable an easy prediction of 3D fibrin network microstructure by the fibril formation kinetic and contribute to an improved engineering of defined scaffolds for tissue engineering and cell culture applications.

Hyaluronan and Collagen Are Prominent Extracellular Matrix Components in Bovine and Porcine Ovaries.

The extracellular matrix (ECM) is a major component of the ovarian stroma. Collagen and hyaluronan (HA) are critical ovarian stromal ECM molecules that undergo age-dependent changes in the mouse and human. How these matrix components are regulated and organized in other mammalian species with reproductive characteristics similar to women such as cows and pigs, has not been systematically investigated. Therefore, we performed histological, molecular, and biochemical analyses to characterize collagen and HA in these animals. Bovine ovaries had more collagen than porcine ovaries when assessed biochemically, and this was associated with species-specific differences in collagen gene transcripts: Col3a1 was predominant in cow ovaries while Col1a1 was predominant in pig ovaries. We also observed more HA in the porcine vs. bovine ovary. HA was distributed across three molecular weight ranges (<100 kDa, 100–300 kDa, and >300 kDa) in ovarian tissue and follicular fluid, with tissue having more >300 kDa HA than the other two ranges. Transcripts for HA synthesis and degradation enzymes, Has3 and Hyal2, respectively, were predominant in cow ovaries, whereas Has2, Kiaa1199, and Tmem2 tended to be predominant in pig ovaries. Together, our findings have implications for the composition, organization, and regulation of the ovarian ECM in large mammalian species, including humans.

Loss of phosphatidylserine flippase β-subunit Tmem30a in podocytes leads to albuminuria and glomerulosclerosis.

ABSTRACTThe asymmetric distribution of phosphatidylserine (PS) in the cytoplasmic leaflet of eukaryotic cell plasma membranes is regulated by a group of P4-ATPases (named PS flippases) and the β-subunit TMEM30A. Podocytes in the glomerulus form a filtration barrier to prevent the traversing of large cellular elements and macromolecules from the blood into the urinary space. Damage to podocytes can disrupt the filtration barrier and lead to proteinuria and podocytopathy. We observed reduced TMEM30A expression in patients with minimal change disease and membranous nephropathy, indicating potential roles of TMEM30A in podocytopathy. To investigate the role of Tmem30a in the kidney, we generated a podocyte-specific Tmem30a knockout (KO) mouse model using the NPHS2-Cre line. Tmem30a KO mice displayed albuminuria, podocyte degeneration, mesangial cell proliferation with prominent extracellular matrix accumulation and eventual progression to focal segmental glomerulosclerosis. Our data demonstrate a critical role of Tmem30a in maintaining podocyte survival and glomerular filtration barrier integrity. Understanding the dynamic regulation of the PS distribution in the glomerulus provides a unique perspective to pinpointing the mechanism of podocyte damage and potential therapeutic targets.

Mechanism of Peripheral Nerve Regeneration Using a Bio 3D Conduit Derived from Normal Human Dermal Fibroblasts.

We previously reported the development of a scaffold-free Bio three-dimensional (3D) nerve conduit from normal human dermal fibroblasts (NHDFs). The aim of this study was to investigate the regenerative mechanism of peripheral nerve cells using a Bio 3D conduit in a rat sciatic nerve defect model. Bio 3D conduits composed of NHDFs were developed, and cell viability was evaluated using a LIVE/DEAD cell viability assay immediately before transplantation and 1-week post-surgery. Tracking analysis using PKH26-labeled NHDFs was performed to assess the distribution of NHDFs within the regenerated nerve and the differentiation of NHDFs into functional Schwann cells (SCs). The assessment of the viability of cells within the Bio 3D conduit showed high cell viability both immediately before transplantation and 1-week post-surgery (88.56 ± 1.70 and 87.58 ± 9.11, respectively). A modified Masson's trichrome staining of the Bio 3D conduit revealed the formation of a prominent extracellular matrix (ECM) in between the cells. We observed, via tracking analysis, that the tube-like distribution of the NHDFs remained stable, the majority of the regenerated axons had penetrated this structure and PKH26-labeled cells were also positive for S-100. Abundant ECM formation resulted in a stable tube-like structure of the Bio 3D conduit with high cell viability. NHDFs in the Bio 3D conduit have the potential to differentiate into SCs-like cells.

Abstract 3947: Mass-spectrometry analysis of metastatic matrisome from MC38 experimental liver metastasis

Abstract Prognosis of patients with colorectal cancer liver metastasis is generally dismal due to unavailability of effective therapeutic modalities. Thus, more basic research is required to extend our knowledge about mechanisms underlying development of hepatic metastasis. The extracellular matrix (ECM) represents a backbone scaffold supporting tissue integrity and establishing cell-cell communications; tumor stroma is highly enriched in certain ECM components which have been suggested as drivers of metastatic dissemination. We performed a label-free quantitative mass-spectrometry (MS) analysis of the ECM isolated from experimental liver metastasis generated by intrasplenic injection of MC38 colon cancer cells. As a group of comparison, we used unaffected mouse liver tissue. Extraction of the ECM was performed utilizing an original method of detergent decellularization, fractionation, followed by biochemical enrichment (i.e. depletion of polysaccharides and nucleic acids) developed in our lab. Label-free MS analysis of resultant ECM samples revealed 2328 proteins, of which 140 were classified as murine matrisome components. Inter-sample variability between ECM molecules was insignificant. Among 140 identified ECM molecules, 13 matrix proteins were dramatically upregulated in metastasis tissues in comparison with tumor-free liver ECM. A combination of these 13 ECM proteins significantly predicted overall survival from colorectal adenocarcinoma based on assessment of 486 patients (P&amp;lt;0.01). One of the most prominent ECM candidates was Annexin A1 (ANXA1), an ECM-affiliated protein consistently overrepresented in the murine metastatic tissue by 7 fold with the highest significance score (P&amp;lt;0.0001). We further validated ANXA1 overexpression in human hepatic metastasis tissues. Higher concentrations of tumor ANXA1 tended to predict colorectal cancer prognosis with borderline significance (P=0.06). Using immunohistochemistry, we identified major sources of ANXA1 in the tumor microenvironment, which were the cancer cells, myeloid cells and T cells. ANXA1 has been previously linked to chemoresistance and altered TGF-beta signaling in cancer cells. To explore the functional role of ANXA1, we performed a stable knockdown of this molecule using shRNA transfection. In vitro, ANXA1-deficient cancer cells displayed slower growth rates but elevated migratory capabilities. In vivo, ANXA1 knockdown cells grew significantly slower as compared to vector control cells when injected subcutaneously, suggesting its role in tumor outgrowth. To conclude, here were report a comprehensive proteomics-aided ECM characterization of MC38 murine colorectal liver metastasis and suggest ANXA1 as a possible target for this disease. Citation Format: Arseniy E. Yuzhalin, Dihua Yu, Ruth J. Muschel. Mass-spectrometry analysis of metastatic matrisome from MC38 experimental liver metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3947.

Proteomic analyses of ECM during pancreatic ductal adenocarcinoma progression reveal different contributions by tumor and stromal cells

Pancreatic ductal adenocarcinoma (PDAC) has prominent extracellular matrix (ECM) that compromises treatments yet cannot be nonselectively disrupted without adverse consequences. ECM of PDAC, despite the recognition of its importance, has not been comprehensively studied in patients. In this study, we used quantitative mass spectrometry (MS)-based proteomics to characterize ECM proteins in normal pancreas and pancreatic intraepithelial neoplasia (PanIN)- and PDAC-bearing pancreas from both human patients and mouse genetic models, as well as chronic pancreatitis patient samples. We describe detailed changes in both abundance and complexity of matrisome proteins in the course of PDAC progression. We reveal an early up-regulated group of matrisome proteins in PanIN, which are further up-regulated in PDAC, and we uncover notable similarities in matrix changes between pancreatitis and PDAC. We further assigned cellular origins to matrisome proteins by performing MS on multiple lines of human-to-mouse xenograft tumors. We found that, although stromal cells produce over 90% of the ECM mass, elevated levels of ECM proteins derived from the tumor cells, but not those produced exclusively by stromal cells, tend to correlate with poor patient survival. Furthermore, distinct pathways were implicated in regulating expression of matrisome proteins in cancer cells and stromal cells. We suggest that, rather than global suppression of ECM production, more precise ECM manipulations, such as targeting tumor-promoting ECM proteins and their regulators in cancer cells, could be more effective therapeutically.

Differential Expression of Extracellular Matrix Components in Nasal Polyp Endotypes.

Background Chronic rhinosinusitis is a difficult-to-treat disease that is often characterized by recurrent nasal polyp (NP) growth following surgical removal. The disease has been separated into distinct phenotypes based on cellular infiltrate or underlying physiological mechanisms. NPs are composed in part of an inflammatory cellular infiltrate, blood vessels, and a large amount of extracellular matrix (ECM). Despite the recognition of prominent ECM deposition, few studies have examined the components in detail and how they might differ with disease state. Objective The purpose of this study was to quantitate the expression of ECM components in NPs. Methods NPs were stained with pico-sirius red to determine total collagen content, and immunofluorescence was used to detect collagen I, collagen III, collagen IV, fibronectin, and laminin. Expression of each was quantitated and analyzed in relation to rhinosinusitis phenotype and separately as a function of polyp eosinophil number. Results When analyzed by phenotype, collagen I, collagen III, and fibronectin were expressed at the highest levels in noneosinophilic sinus disease. Collagen IV was not different among any groups, and its location was found predominately around vessels. When analyzed as a function of polyp eosinophil number, total collagen and collagen III showed a significant inverse correlation. Conclusions NP ECM composition differs with disease state with higher expression in cases where eosinophil levels are low. This suggests that in eosinophilic polyps there is a loss of matrix deposition either through break down or a failure to produce the essential components. Understanding these differences may identify new therapeutic targets.

Impact of several proinflammatory and cell degradation factors in patients with aortic valve stenosis.

Aortic valve (AoV) stenosis is the third most common cardiovascular disease. The pathogenesis of AoV stenosis is associated with an inflammatory process where MMPs serve important roles. The aim of the present study was to determine the association between matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and inflammatory factors, and AoV stenosis at various degrees of severity compared with the control. A total of 18 patients with mild, 19 with moderate and 15 with severe AoV stenosis were included in the present stud, and 50 individuals were enrolled in the control group. The severity of stenosis was determined by echocardiography. The expression levels of chemerin, fibroblast growth factor 21, MMP-1, −3, and −9, and TIMP-1 and −3 were analyzed by ELISA. Data were analyzed using GraphPad Prism7 software. The expression levels of MMP-1 was increased in patients with stenosis compared with the control group (P=0.0043). Distribution of the trimodal MMP-1 values was obtained in the stenosis group and monomodal in the control group. A total of 80% of patients in the stenosis group presented significantly increased expression levels of MMP-1 compared with the control group (P=0.0002). Expression of MMP-1 was significantly higher in all stenosis groups compared with the control. The highest expression level of MMP-1 appeared in patients with moderate stenosis (P<0.0001). There was no significant difference in the expression of MMP-3, MMP-9 and TIMP-1 in the aortic stenosis group, compared with the control group. A positive correlation between MMP-1 and MMP-9 expression levels was identified (r=0.37; P=0.017). The increase of MMP-1 was correlated with the increase of MMP-9, but not with the level of MMP-3. The expression levels of chemerin was significantly elevated in patients with stenosis compared with healthy patients. The highest expression levels of chemerin were determined in patients with mild (P=0.0001) and moderate (P=0.0007) stenosis and decreased with the grade of severity compared with the control group. The expression of FGF-21 was significantly different between the control and mild (P=0.013), moderate (P=0.015) and severe stenosis (P=0.003) groups. The expression levels of FGF-21 increased with the increase in severity grade, reaching the maximum for severe stenosis. The results of the present study indicated that the inflammatory process is predominantly occurring at the early, mild stage of stenosis and the most prominent extracellular matrix remodeling occurs in moderate stenosis (demonstrated by MMP-1 levels). In patients with severe stenosis, the levels of MMP-1 and chemerin (which are lower than in a case of mild or moderate stenosis) could indicate the development of calcinosis and the reduction in activity or inactivation of the inflammatory process.

A Review of Integrin-Mediated Endothelial Cell Phenotype in the Design of Cardiovascular Devices.

Sustained biomaterial thromboresistance has long been a goal and challenge in blood-contacting device design. Endothelialization is one of the most successful strategies to achieve long-term thromboresistance of blood-contacting devices, with the endothelial cell layer providing dynamic hemostatic regulation. It is well established that endothelial cell behavior is influenced by interactions with the underlying extracellular matrix (ECM). Numerous researchers have sought to exploit these interactions to generate improved blood-contacting devices by investigating the expression of hemostatic regulators in endothelial cells on various ECM coatings. The ability to select substrates that promote endothelial cell-mediated thromboresistance is crucial to advancing material design strategies to improve cardiovascular device outcomes. This review provides an overview of endothelial cell regulation of hemostasis, the major components found within the cardiovascular basal lamina, and the interactions of endothelial cells with prominent ECM components of the basement membrane. A summary of ECM-mimetic strategies used in cardiovascular devices is provided with a focus on the effects of key adhesion modalities on endothelial cell regulators of hemostasis.

Pirfenidone decreases mesothelioma cell proliferation and migration via inhibition of ERK and AKT and regulates mesothelioma tumor microenvironment in vivo

Malignant mesothelioma is an aggressive cancer with poor prognosis. It is characterized by prominent extracellular matrix, mesenchymal tumor cell phenotypes and chemoresistance. In this study, the ability of pirfenidone to alter mesothelioma cell proliferation and migration as well as mesothelioma tumor microenvironment was evaluated. Pirfenidone is an anti-fibrotic drug used in the treatment of idiopathic pulmonary fibrosis and has also anti-proliferative activities. Mesothelioma cell proliferation was decreased by pirfenidone alone or in combination with cisplatin. Pirfenidone also decreased significantly Transwell migration/invasion and 3D collagen invasion. This was associated with increased BMP pathway activity, decreased GREM1 expression and downregulation of MAPK/ERK and AKT/mTOR signaling. The canonical Smad-mediated TGF-β signaling was not affected by pirfenidone. However, pirfenidone blocked TGF-β induced upregulation of ERK and AKT pathways. Treatment of mice harboring mesothelioma xenografts with pirfenidone alone did not reduce tumor proliferation in vivo. However, pirfenidone modified the tumor microenvironment by reducing the expression of extracellular matrix associated genes. In addition, GREM1 expression was downregulated by pirfenidone in vivo. By reducing two major upregulated pathways in mesothelioma and by targeting tumor cells and the microenvironment pirfenidone may present a novel anti-fibrotic and anti-cancer adjuvant therapy for mesothelioma.

Identification of Mature Atherosclerotic Plaque Proteome Signatures Using Data-Independent Acquisition Mass Spectrometry.

Atherosclerosis is a chronic inflammatory disease with complex pathobiology and one of the most common causes of cardiovascular events. The process is characterized by complex vascular remodeling processes that require the actions of numerous proteins. The composition of atherosclerotic plaque is increasingly recognized as a major factor governing the occurrence of cardiovascular or neurological symptoms. To gain deeper insights into the composition of atherosclerotic plaques, we created quantitative proteome profiles of advanced plaque tissues of six male patients undergoing carotid endarterectomy for stroke prevention. Using a quantitative, data-independent proteome approach, we identified 4181 proteins with an average protein coverage of 45%. An analysis of the quantitative composition of the tissue revealed key players of vascular remodeling processes. Moreover, compared with proximal arterial tissue, 20 proteins in mature plaques were enriched, whereas 52 proteins were found in lower quantities. Among the proteins with increased abundance were prominent extracellular matrix proteins such as biglycan and lumican, whereas cytoskeletal markers for contractile smooth muscle cells (SMCs) were decreased. Taken together, this study provides the most comprehensive quantitative assessment of mature human plaque tissue to date, which indicates a central role of SMCs in the structure of advanced atherosclerotic plaques.

Lysophosphatidic acid-induced vascular neointimal formation in mouse carotid arteries is mediated by the matricellular protein CCN1/Cyr61.

Vascular smooth muscle cell (SMC) migration is an essential step involved in neointimal formation in restenosis and atherosclerosis. Lysophosphatidic acid (LPA) is a bioactive component of oxidized low-density lipoprotein and is produced by activated platelets, implying that LPA influences vascular remodeling. Our previous study revealed that matricellular protein CCN1, a prominent extracellular matrix (ECM) protein, mediates LPA-induced SMC migration in vitro. Here we examined the role of CCN1 in LPA-induced neointimal formation. By using LPA infusion of carotid artery in a mouse model, we demonstrated that LPA highly induced CCN1 expression (approximately six- to sevenfold) in neointimal lesions. Downregulation of CCN1 expression with the specific CCN1 siRNA in carotid arteries blocked LPA-induced neointimal formation, indicating that CCN1 is essential in LPA-induced neointimal formation. We then used LPA receptor knockout (LPA1-/-, LPA2-/-, and LPA3-/-) mice to examine LPA receptor function in CCN1 expression in vivo and in LPA-induced neointimal formation. Our data reveal that LPA1 deficiency, but not LPA2 or LPA3 deficiency, prevents LPA-induced CCN1 expression in vivo in mouse carotid arteries. We also observed that LPA1 deficiency blunted LPA infusion-induced neointimal formation, indicating that LPA1 is the major mediator for LPA-induced vascular remodeling. Our in vivo model of LPA-induced neointimal formation established a key role of the ECM protein CCN1 in mediating LPA-induced neointimal formation. Our data support the notion that the LPA1-CCN1 axis may be the central control for SMC migration and vascular remodeling. CCN1 may serve as an important vascular disease marker and potential target for vascular therapeutic intervention.

In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy

Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to abnormal matrix production and tissue invasion contributing to the development of the disease. In this study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins, 17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis showed that these proteins either belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding. Further pathway analysis revealed that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

Abstract 163: Delayed Administration of Perlecan Doman V Significantly Increases Neurogenesis and Improves Functional Outcome after Experimental Ischemic Stroke

Stroke, a major cause of significant morbidity and death, has limited therapeutic options. To that end, our goal is to develop novel stroke therapies by exploiting the brain’s own neuroreparative potential. We have discovered that perlecan, a prominent brain extracellular matrix proteoglycan, is proteolyzed into the bioactive protein fragment domain V (DV) after experimental and human ischemic stroke. DV is both neuroprotective and pro-angiogenic. Furthermore, functional outcome can be significantly improved in rodents by administering DV 6-24 hours after experimental transient (distal MCA occlusion) or permanent (photothrombosis) stroke. Here we studied in mechanistic detail whether delayed DV administration (initiated 7 days post-stroke) could increase neurogenesis and improve functional outcome after mouse transient MCA occlusion. Delayed DV administration also served to minimize confounding neuroprotective and angiogenic effects which occur with acute DV therapy. After two doses of DV (post-stroke day 7 and 9), stroked 3-month old male C57BL6 mice had significant functional improvement (rotor rod) compared to vehicle treated stroke controls. Brain immunohistochemistry 21 days after stroke demonstrated that DV treated mice had significantly more cells that were positive for BrdU (a marker of cell division), doublecortin (DCX, a marker for immature neurons), and NeuN (another neuronal marker) in the infarct area. Furthermore, stereotactic brain injection (AP + 0.00, ML - 1.2, DV – 2.5) with alpha 2 integrin (a known neuronal DV receptor) function blocking antibody on post-stroke day 3 inhibited DV’s post-stroke neurogenic effects. These results suggest that delayed DV treatment after experimental stroke increases neurogenesis, increases the number of new neurons that reach stroked brain regions and survive there, and improves functional outcome through an alpha 2 integrin dependent mechanism. Importantly, as we are unaware of any other delayed treatment other than FLAME clinical trial (fluoxetine treatment initiated 5-10 days after stroke) that significantly improves functional outcome after stroke, our data further suggest the promise of DV as an effective stroke therapy.

Time-Dependent Extracellular Matrix Organization and Secretion from Vascular Endothelial Cells due to Macromolecular Crowding

ABSTRACTInteractions between biological cells and surrounding extracellular matrix (ECM) materials modulate many cell behaviors including adhesion and migration. One key example of this cellmatrix reciprocity is in the context of angiogenesis, the sprouting of new blood vessels from preexisting vasculature. Vascular endothelial cells (VECs) create and remodel the ECM during this process. In vivo, the surrounding fluid environment includes high concentrations of macromolecules, and is considered “crowded” in comparison to in vitro environments. Here, we quantified the amount and organization of collagen IV, a prominent ECM component of VECs, that was produced by these cells over four weeks in vitro in the presence or absence of macromolecular crowder (MMC) nanoparticles that approximated in vivo crowding. In the presence of MMCs, the amount and degree of alignment of collagen IV was greater. This ECM difference emerged within one week and was sustained for over four weeks. We explored the effect of initial cell density (cells/µm2) on this matrix production, to consider potential differences at a wound site versus an intact vessel. Moreover, we found the biophysical effect of MMCs to be unmodulated by secretions from an adjacent cell type in microvessels (pericytes). These results suggest that macromolecular crowding plays a direct role in remodeling the basement membrane, and that such crowding can be induced in vitro to more closely approximate the cell microenvironment.

Translating an Understanding of the Pathogenesis of Hepatic Fibrosis to Novel Therapies

The response to injury is one of wound healing and fibrogenesis, which ultimately leads to fibrosis. The fibrogenic response to injury is a generalized one across virtually all organ systems. In the liver, the injury response, typically occurring over a prolonged period of time, leads to cirrhosis (although it should be pointed out that not all patients with liver injury develop cirrhosis). The fact that many different diseases result in cirrhosis suggests a common pathogenesis. The study of hepatic fibrogenesis over the past 2 decades has been remarkably active, leading to a considerable understanding of this process. It clearly has been shown that the hepatic stellate cell is a central component in the fibrogenic process. It also has been recognized that other effector cells are important in the fibrogenic process, including resident fibroblasts, bone marrow-derived cells, fibrocytes, and even perhaps cells derived from epithelial cells (ie, through epithelial to mesenchymal transition). A key aspect of the biology of fibrogenesis is that the fibrogenic process is dynamic; thus, even advanced fibrosis (or cirrhosis) is reversible. Together, an understanding of the cellular basis for liver fibrogenesis, along with multiple aspects of the basic pathogenesis of fibrosis, have highlighted many exciting potential therapeutic opportunities. Thus, although the most effective antifibrotic therapy is simply treatment of the underlying disease, in situations in which this is not possible, specific antifibrotic therapy is likely not only to become feasible, but will soon become a reality. This review highlights the mechanisms underlying fibrogenesis that may be translated into future antifibrotic therapies and to review the current state of clinical development.

Force-induced fibronectin assembly and matrix remodeling in a 3D microtissue model of tissue morphogenesis

Encapsulations of cells in type-I collagen matrices are widely used three-dimensional (3D) in vitro models of wound healing and tissue morphogenesis and are common constructs for drug delivery and for in vivo implantation. As cells remodel the exogenous collagen scaffold, they also assemble a dense fibronectin (Fn) matrix that aids in tissue compaction; however, the spatio-temporal (re)organization of Fn and collagen in this setting has yet to be quantitatively investigated. Here, we utilized microfabricated tissue gauges (μTUGs) to guide the contraction of microscale encapsulations of fibroblasts within collagen gels. We combined this system with a Foerster Radius Energy Transfer (FRET) labeled biosensor of Fn conformation to probe the organization, conformation and remodeling of both the exogenous collagen and the cell-assembled Fn matrices. We show that within hours, compact Fn from culture media adsorbed to the collagen scaffold. Over the course of tissue remodeling, this Fn-coated collagen scaffold was compacted into a thin, sparsely populated core around which cells assembled a dense fibrillar Fn shell that was rich in both cell and plasma derived Fn. This resulted in two separate Fn populations with different conformations (compact/adsorbed and extended/fibrillar) in microtissues. Cell contractility and microtissue geometry cooperated to remodel these two populations, resulting in spatial gradients in Fn conformation. Together, these results highlight an important spatio-temporal interplay between two prominent extracellular matrix (ECM) molecules (Fn and collagen) and cellular traction forces, and will have implications for future studies of the force-mediated remodeling events that occur within collagen scaffolds either in 3D in vitro models or within surgical implants in vivo.

Secretion of collagen I and tenascin is modulated by laminin‐111 in 3D culture of human adenoid cystic carcinoma cells

Adenoid cystic carcinoma is a frequent malignant salivary gland neoplasm with high levels of recurrence and metastasis. This neoplasm expresses prominent extracellular matrix (ECM). We are studying regulatory mechanisms underlying secretion of ECM molecules in adenoid cystic carcinoma. We have previously demonstrated that laminin modulates the phenotype of a human adenoid cystic carcinoma (CAC2) cell line. Thus, this molecule would be a good candidate to regulate secretion of ECM molecules in these cells. Here we analysed the role played by laminin-111 [formerly laminin-1; Aumailley et al. (2005). Matrix Biol. 24, 326] stimulating secretory activity of CAC2 cells. Three-dimensional cultures of cells in laminin-111 (treated) or agarose (controls) were studied by light and electron microscopy. Ultrastructural analysis of CAC2 cells grown within laminin-111 showed pseudocysts filled with secretory-like material. Cells exhibited prominent and dilated endoplasmic reticulum and coated and uncoated vesicles. Ultrastructural findings suggested that laminin-111 induced secretory activity in CAC2 cells. We further investigated this point by light microscopy, immunofluorescence and confocal microscopy. Histochemistry showed periodic acid-Schiff (PAS)-positive diastase-resistant material in CAC2 cells treated by laminin-111. This material could represent laminin-induced secretion of ECM molecules. We searched for collagen I and tenascin in CAC2 cells treated by laminin-111. Confocal microscopy and immunoblot showed that laminin-111 enhanced secretion of collagen I and tenascin in CAC2 cells. We suggest that laminin-111 modulates secretion of collagen I and tenascin in cells derived from human adenoid cystic carcinoma.