Purified Extracellular Matrix Proteins Research Articles

Extracellular vesicles from non-neuroendocrine SCLC cells promote adhesion and survival of neuroendocrine SCLC cells.

Small cell lung cancer (SCLC) tumors are made up of distinct cell subpopulations, including neuroendocrine (NE) and non-neuroendocrine (non-NE) cells. While secreted factors from non-NE SCLC cells have been shown to support the growth of the NE cells, the underlying molecular factors are not well understood. Here, we show that exosome-type small extracellular vesicles (SEVs) secreted from non-NE SCLC cells promote adhesion and survival of NE SCLC cells. Proteomic analysis of purified SEVs revealed that extracellular matrix (ECM) proteins and integrins are highly enriched in SEVs of non-NE cells whereas nucleic acid-binding proteins are enriched in SEVs purified from NE cells. Addition of select purified ECM proteins identified in purified extracellular vesicles (EVs), specifically fibronectin, laminin 411, and laminin 511, were able to substitute for the role of non-NE-derived SEVs in promoting adhesion and survival of NE SCLC cells. Those same proteins were differentially expressed by human SCLC subtypes. These data suggest that ECM-carrying SEVs secreted by non-NE cells play a key role in supporting the growth and survival of NE SCLC cells.

Differentiating Human Pluripotent Stem Cells to Cardiomyocytes Using Purified Extracellular Matrix Proteins.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.

Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP.RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide. Statement of SignificancePancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.

Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array

The brain’s extracellular matrix (ECM) is a macromolecular network composed of glycosaminoglycans, proteoglycans, glycoproteins, and fibrous proteins. In vitro studies often use purified ECM proteins for cell culture coatings, however these may not represent the molecular complexity and heterogeneity of the brain’s ECM. To address this, we compared neural network activity (over 30 days in vitro) from primary neurons co-cultured with glia grown on ECM coatings from decellularized brain tissue (bECM) or MaxGel, a non-tissue-specific ECM. Cells were grown on a multi-electrode array (MEA) to enable noninvasive long-term interrogation of neuronal networks. In general, the presence of ECM accelerated the formation of networks without affecting the inherent network properties. However, specific features of network activity were dependent on the type of ECM: bECM enhanced network activity over a greater region of the MEA whereas MaxGel increased network burst rate associated with robust synaptophysin expression. These differences in network activity were not attributable to cellular composition, glial proliferation, or astrocyte phenotypes, which remained constant across experimental conditions. Collectively, the addition of ECM to neuronal cultures represents a reliable method to accelerate the development of mature neuronal networks, providing a means to enhance throughput for routine evaluation of neurotoxins and novel therapeutics.

Abstract A38: Biophysical Regulation of Breast Cancer Metastasis

Abstract Introduction: Despite many advances in our understanding of breast cancer biology over the last decades, there are no therapies that can effectively prevent or treat metastatic cancer. During the growth of the primary tumor mass and its dissemination through the body during metastasis, cancer cells are exposed to a host of mechanical environments generated by the local matrix compliance, pressure and tension from tumor mass expansion, and fluidic shear stresses from interstitial and vascular fluid flow. These biophysical forces are emerging as powerful regulators of cancer growth, quiescence and metastasis; however, our understanding of the mechanisms of the biomechanical regulation of tumor biology remains very limited. The overall goal was to identify the role of mechanical forces in regulating key steps in tumor metastasis and progression. Results: We recently developed a mesofluidic system that allows the high throughput study of tumor cell interactions under flow. This device allows us to simulate the adhesion of cancer cells to endothelial cells under physiological flow conditions to model this step of the metastatic cascade and to apply shear forces to cells in a 96-well format. In addition, we have designed a system to apply mechanical stretch to cells in a high throughput format (576 wells simultaneously). Using these two systems, we examined the interplay between mechanical cues and the propensity of breast cancer cells to metastasize and undergo epithelial-to-mesenchymal transition (EMT). Mechanical strain increases circulating tumor cell adhesion to endothelial cells and extracellular matrix (ECM). We applied cyclic mechanical strain (5% maximal strain) to MDA-MB-231 and MCF-7 cancer cells for 24 hours and then performed adhesion assays of the cancer cells to activated and non-activated endothelial cells (ECS) and purified ECM proteins. We found that cyclic strain increased cancer cell adhesion to activated ECs in comparison to non-strained cells. In addition cancer cells exposed to mechanical strain adhered more to collagen I, laminin, and vitronectin, while they adhered less to collagen II and fibronectin in comparison to non-strained control cells. To determine which integrins were involved in the strain-induced change in adhesion, we treated the cells with a library of integrin inhibitors while applying strain for 24 hours. Cilengitide, P11, ATN-161, Bio 1211, and RGDS peptides reduced the adhesion of cancer cells back to the level of non-strained cancer cells, indicating the role of αvβ3, αvβ5, α5β1, and α4β1 integrins in the biophysical regulation of circulating tumor cell adhesion. Cyclic strain alters the ability of breast cancer cells to invade through an endothelial monolayer. We next used the high throughput system to apply multiple levels of strain to the cancer cell lines (0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5% strain). We then trypsinized the cells and measured their invasion in a Transwell assay with a confluent layer of endothelial cells cultured on a porous membrane. We found that the mid-level strains ranging from 7.5-15% strain decreased the invasiveness of the MDA-MB-231 cells compared to the non-strained control cells. For the MCF-7 cells, mechanical strain of 5% or higher led to decreased invasion through the endothelial layer. Mechanical forces alter signaling through the TGF-β and Yap/Taz pathways as well the expression of markers of epithelial-to-mesenchymal transition (EMT). We applied cyclic strain at multiple levels (2.5-17.5% strain) to breast cancer cells for 24 hours then immunostained for various signaling proteins. In MDA-MB-231 cells, we saw a significant decrease in the intensity of Smad2/3 and nuclear phospho-Smad2/3 at mid-range strains (5-12.5% strain) as well as an increase in the nuclear localization of Yap/Taz using fluorescent immunostaining. At high levels of strain (15 and 17.5% strain), there was increased nuclear p-Smad2/3 intensity. Consistent with these findings, we found increased Smad activity in the cells using a luciferase reporter assay. An analysis of EMT markers using PCR, western blotting and immunostaining showed a reduction in mesenchymal markers including α−SMA, Slug, ZEB, fibronectin and vimentin with mid-range mechanical strain levels. Conclusion: Together, our studies demonstrate that mechanical forces can profoundly alter the propensity of cancer cells to adhere and invade during metastasis. Moreover, the response to mechanical strain is dependent on the magnitude of the strain and cancer cell type. These effects are mediated, in part, through integrin interactions and the differential regulation of the TGF-β and Hippo pathways by biophysical forces. Citation Format: Adrianne Spencer, Jason Lee, Katerina Lee, Darshil Choksi, Jerry Wang, Christopher Spruell, Aaron Baker. Biophysical Regulation of Breast Cancer Metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A38.

Harnessing cellular-derived forces in self-assembled microtissues to control the synthesis and alignment of ECM

The alignment and blend of extracellular matrix (ECM) proteins give a tissue its specific mechanical properties as well as its physiological function. Various tissue engineering methods have taken purified ECM proteins and aligned them into gels, sponges and threads. Although, each of these methods has created aligned ECM, they have had many limitations including loss of hierarchal collagen structure and poor mechanical performance. Here, we have developed a new method to control ECM synthesis using self-assembled cells. Cells were seeded into custom designed, scaffold-free, micro-molds with fixed obstacles that harnessed and directed cell-mediated stresses. Cells within the microtissue reacted to self-generated tension by aligning, elongating, and synthesizing an ECM whose organization was dictated by the strain field that was set by our micro-mold design. We have shown that through cell selection, we can create tissues with aligned collagen II or aligned elastin. We have also demonstrated that these self-assembled microtissues have mechanical properties in the range of natural tissues and that mold design can be used to further tailor these mechanical properties.

Abstract 15972: Fibronectin is Essential for Mesendoderm Induction During Cardiac Differentiation of Human Pluripotent Stem Cells

Extracellular matrix (ECM) plays important roles in development, and we have previously demonstrated a matrix sandwich protocol for efficient cardiac differentiation of human pluripotent stem cells (hPSCs). The purpose of the present study was to identify the key ECM proteins required to initiate cardiogenic differentiation of hPSCs and define their mechanism of action. We tested purified ECM proteins including human laminin (LN111, LN521), fibronectin (FN) and collagen IV in the matrix sandwich protocol, and found FN promoted efficient cardiac differentiation. To determine if FN is essential for the early stages of cardiogenesis we tested three FN blockers: FUD peptide that binds the N-terminal 70-kDa fragment of FN, anti-human FN antibody clone10 which recognizes FN domain III2, and anti-human FN antibody clone 3E3 that binds on the cell attachment domain. All three blockers showed concentration-dependent inhibition of cardiac differentiation of hPSCs when added at day 0 for 24 hours, measured by flow cytometry for cTnT+ cells in the Matrigel/Matrigel (bottom/overlay) and FN/FN sandwich culture as well as in the small molecule GiWi protocol. Furthermore, clone10 showed significant inhibition of cardiac differentiation even in the absence of adding exogenous FN due to the robust endogenous expression of FN by the hPSCs. Gene expression analysis with qRT-PCR demonstrated that blocking FN by clone10 caused a significant suppression of gene expression related to: 1) epithelial to mesenchymal transition, e.g. SNAIL1, SNAIL2, GSC, VIM, CDH2 and FN1; 2) Mesendoderm e.g. T, MIXL1, SOX17 and precardiac mesoderm of MESP1; 3) Cardiac transcription factors ISL1 and NKX2.5. To investigate the cellular signaling activated by FN, we tested a blocking antibody (clone P5D2) to the major FN receptor, integrin β1, and found that P5D2 produced a concentration-dependent inhibition of cardiogenesis in multiple hPSC lines. Furthermore, the integrin-linked kinase (ILK) inhibitor, Cpd22, inhibited cardiac differentiation in a concentration-dependent fashion as well when added on day 0. We conclude that FN, acting via integrin β1-activated ILK signaling, is essential to mesendoderm formation enabling cardiogenesis of hPSCs.

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix

BackgroundMyeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. MethodsWe investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. ResultsMPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. ConclusionsCollectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. General significanceOur findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age-related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC-RPEs) and iPSCs (iPSC-RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell-RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC-RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin-111 supporting the highest pigmentation frequencies. Although iPSC-RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin-111. iPSCs differentiated on mouse laminin-111 produced iPSC-RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin-111 resulted in high yields of functional hESC-RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno-free, GMP-compliant generation of RPE from human pluripotent stem cells relevant to clinical translation.

Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin.

A PSP94-derived Peptide PCK3145 inhibits MMP-9 Secretion and Triggers CD44 Cell Surface Shedding: Implication in Tumor Metastasis

PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor.

Differential Pattern of Integrin Receptor Expression in Differentiated and Anaplastic Thyroid Cancer Cell Lines

Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of thyroid tumor cell lines to ECM may not be associated with altered IRM expression.

Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus invade the extracellular matrix (ECM) using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Normal cells neither form invadopodia nor degrade the ECM. Here we show that cells expressing invadopodia degrade and enter into a fibronectin-rich matrix produced by normal fibroblasts. Within 6 h after seeding onto the matrix, the invasive cells create an area devoid of matrix fibrils surrounding the cell body. Proteolysis mediates this matrix clearing because sevenfold more radiolabeled matrix is released into the growth media by the transformed cells relative to the normal cells. In addition to this assembled matrix, transformed cells were grown on thin layers of purified ECM proteins, revealing that invadopodia can degrade fibronectin, collagen type I, collagen type IV, and laminin. A 160 kDa protease that is extracted from transformed cells by Triton X-114 partitions into the detergent phase and is prominent in ventral plasma membranes that contact the ECM suggesting that it is a membrane associated protease.

Interferon-Gamma and Tumor Necrosis Factor-Alpha Promote the Binding of Dendritic Cells to Fibronectin

Connective tissue dendritic accessory cells (DC) accumulate at sites of extracellular matrix (ECM) deposition. The adherence of DC to purified ECM proteins was examined. Splenic and pulmonary DC were purified from Lewis rats and incubated on slides coated with fibronectin (FN), laminin (LN), or collagen (COLL) types I, III and IV. In other experiments, DC were pre-incubated with selected proinflammatory cytokines (IL-1, IL-2, IFN-gamma, TNF-alpha) in order to determine their abilities to modulate cell adherence to ECM. Both splenic and pulmonary DC showed dose-dependent binding to FN that was inhibited by the synthetic peptide arg-gly-asp-ser. There was minimal DC binding to LN or COLL. Pretreating DC with IFN-gamma or TNF-alpha enhanced DC binding to FN but did not increase binding to LN or COLL. IL-1 and IL-2 had no demonstrable effect on DC binding to ECM. Our results suggest that FN is a critical ligand for DC in their binding to the ECM. IFN-gamma and TNF-alpha increase adherence of DC to FN and may promote their accumulation in the lung during inflammation.

Adhesion of rat glomerular epithelial cells to extracellular matrices: Role of β1 integrins

Glomerular epithelial cells (GEC) maintain glomerular permselectivity and are a target of immunological glomerular injury, which may lead to proliferation or detachment from extracellular matrix (ECM). We studied adhesion mechanisms in rat GEC in culture, focusing on adhesion molecules of the beta 1 integrin family. At early time points (1 hr after plating of cells into culture wells that had been pre-incubated with purified ECM proteins), adhesion of GEC to collagen I, collagen IV, laminin, and fibronectin was inhibited with anti-beta 1 integrin antibody. The peptide RGDS inhibited adhesion to fibronectin and laminin. Immunoprecipitation studies demonstrated the presence of alpha 2, alpha 3, and beta 1 integrins; the alpha 1, alpha 4, alpha 5, alpha 6, alpha v, and beta 3 subunits were undetectable. Adhesion to all ECM proteins was dependent on divalent cations, but the effects of individual cations varied among substrata. In rat GEC, alpha 2 beta 1 and/or alpha 3 beta 1 integrins appear to mediate adhesion to collagen I, collagen IV, and laminin. The alpha 3 beta 1 integrin is also the likely receptor for fibronectin, interacting through an RGD binding site. Furthermore, single integrins or combinations of integrins appear to have distinct ligand-binding functions that are differentially regulated by divalent cations. Characterization of GEC adhesion molecules may facilitate the understanding of mechanisms of glomerular development, and cell detachment or proliferation in immune glomerular injury.

Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells

The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin>collagen I≥collagen IV≥vitronectin>laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells.