Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix

To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.     

Function at the junction: dynamic interactions between lung cells and extracellular matrix.

Recent advances are elucidating the mechanisms by which cells communicate with the surrounding matrix. Cells have specific receptors for matrix proteins. A number of intracellular molecules with signalling functions aggregate at specialised focal adhesion points and facilitate transfer of information both into and out of cells. The importance of these signaling processes to cell biology makes it likely that manipulation of these processes will allow innovative therapeutic approaches to lung disease.     

Peritoneal mesothelial cells and the extracellular matrix

Continuous ambulatory peritoneal dialysis (CAPD) is an important treatment for patients with end‐stage renal failure. Long‐term success is dependent on the functional and structural integrity of the peritoneal membrane. Conventional peritoneal dialysis fluids are non‐physiological. They contain glucose at high concentrations to provide the osmotic drive for ultrafiltration, lactate to correct the metabolic acidosis of renal failure, and a low pH to prevent caramelization of glucose during heat sterilization. These components, in isolation or acting together, exert adverse influences on both the resident cellular and extracellular elements of the peritoneal membrane, as well as phagocytic cells which infiltrate the peritoneum during inflammation, culminating in detrimental structural and functional effects, compromising the viability of the peritoneum during dialysis. Peritoneal biopsy studies of patients on long‐term CAPD have demonstrated an intercellular space between adjacent mesothelial cells which allows the penetration of peritoneal dialysis fluid into the underlying submesothelium. This, together with episodes of peritonitis, can initiate a chronic inflammatory reaction within the peritoneum characterized by increased synthesis of matrix proteins. Perturbation of the regulatory mechanisms which govern the balance of synthesis and degradation of extracellular matrix can lead to progressive fibrosis. Human peritoneal mesothelial cells (HPMC) have been shown to synthesize fibronectin, laminin, collagens, proteoglycans and hyaluronan in vitro, and thus play a role in the pathogenesis of peritoneal fibrosis. This review will give an overview of extracellular matrix (ECM) synthesis by HPMC, how changes in the synthesis are affected by CAPD and postulate how these changes can compromise the dialytic properties of the peritoneum.     

Antigen‐dependent interactions between regulatory B cells and T cells at the T:B border inhibit subsequent T cell interactions with DCs

IL‐10+ regulatory B cells (Bregs) inhibit immune responses in various settings. While Bregs appear to inhibit inflammatory cytokine expression by CD4+ T cells and innate immune cells, their reported impact on CD8+ T cells is contradictory. Moreover, it remains unclear which effects of Bregs are direct versus indirect. Finally, the subanatomical localization of Breg suppressive function and the nature of their intercellular interactions remain unknown. Using novel tamoxifen‐inducible B cell–specific IL‐10 knockout mice, we found that Bregs inhibit CD8+ T cell proliferation and inhibit inflammatory cytokine expression by both CD4+ and CD8+ T cells. Sort‐purified Bregs from IL‐10‐reporter mice were adoptively transferred into wild‐type hosts and examined by live‐cell imaging. Bregs localized to the T:B border, specifically entered the T cell zone, and made more frequent and longer contacts with both CD4+ and CD8+ T cells than did non‐Bregs. These Breg:T cell interactions were antigen‐specific and reduced subsequent T:DC contacts. Thus, Bregs inhibit T cells through direct cognate interactions that subsequently reduce DC:T cell interactions.     

Human dermal fibroblast subpopulations; differential interactions with vascular endothelial cells in coculture: Nonsoluble factors in the extracellular matrix influence interactions

The superficial dermis of adult human skin contains a complex arcading microvasculature that provides nutrient support to the overlying epidermis. We propose that the unique subpopulations of dermal fibroblasts located in the superficial dermis contribute to the organization and maintenance of this elaborate microvasculature. This possibility was tested in a coculture system in which distinct subpopulations of adult human dermal fibroblasts were grown to form high‐density lawns that were then seeded with human umbilical vein vascular endothelial cells (EC). The fibroblast subpopulation cultured specifically from the papillary dermis supported a robust array of highly branched tube‐like structures. In contrast, fibroblasts cultured from the reticular dermis provided an anemic level of support for the formation of tube‐like structures. These varied interactions with vascular EC were not due to the differential production of the potent pro‐angiogenic factors vascular endothelial growth factor‐A or fibroblast growth factor‐2. Instead, the extracellular matrix and/or molecules bound to this matrix appeared to contain instructions that modulated these differential fibroblast–vascular EC interactions. One matrix‐binding growth factor, hepatocyte growth factor/scatter factor, was identified that was both differentially expressed by papillary and reticular dermal fibroblasts and which was shown to be physiologically relevant in the coculture model. These studies highlight the importance of fibroblasts in supporting and maintaining vascular integrity. Furthermore, these studies have important implications for wound repair and may help to explain how fibroblasts contribute to the etiology of nonhealing wounds.     

Integrin‐mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin‐mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM‐111 and LM‐511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM‐binding integrins was quantified for human NP cells using flow cytometry. The cell‐ECM attachment mechanism was determined by quantifying cell attachment to LM‐111, LM‐511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM‐111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell‐based therapeutics. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1661–1667, 2013     

慢性胰腺炎与细胞外基质代谢失衡的关系

目的　研究慢性胰腺炎与细胞外基质 (ECM )代谢失衡的关系 ,探讨慢性胰腺炎的发病机制。方法　应用含有 40 96条人类全长基因的cDNA表达谱芯片 ,分别对 6例慢性胰腺炎及正常胰腺组织标本的基因表达谱进行分析。结果　在 6例慢性胰腺炎组织中 ,均有差异表达的基因2 0 7条 ,占芯片基因数的 5 .0 1%。从中筛选出与ECM代谢有关的显著表达差异基因 18条 ,其中表达上调基因 10条 ,下调基因 8条。结论　转化生长因子 (TGF) β1持续性高表达造成的ECM代谢紊乱 ,可能是导致慢性胰腺炎胰腺纤维化发生、发展的重要因素。     

Relationship between endothelial cells and extracellular matrix: investigation using the model of angiogenesis in vitro]

Using model of angiogenesis in vitro, the relationship between endothelial cells and extracellular matrix was studied. Endothelial cells of bovine brain microvascular vessels (BBECs), carotid artery (BCECs) and aorta (BAECs) were cultured on type I collagen gel and Matrigel. BBECs make tubular structures and BCECs and BAECs grow and make confluent monolayer on type I collagen gel. BCECs and BAECs make tubular structures when second layer of collagen gel was overlaid. BBECs, BCECs and BAECs rapidly make tubular structures on Matrigel. These morphological changes were not affected by basic fibroblast growth factor. The effect of extracellular matrices on the cell kinetics and morphology of cultured bovine carotid artery endothelial cells was studied. Endothelial cells show a cobble stone appearance on plastic and type I collagen gel. They proliferate and make capillary-like structures on reconstituted gels composed of type I collagen and basement membrane substrata. And that endothelial cells forming capillary-like structures were increased with the increase in the concentration of basement membrane substrata. Transmission electromicroscopic examination study revealed endothelial cells forming capillary-like structures have junctional complexes on type I collagen and its mixture with basement membrane substrata. But endothelial cells on basement membrane substrata have no junctional complexes. These results suggest that BBECs have more potent angiogenic ability than BCECs and BAECs. And that proliferation and morphogenesis of endothelial cells are regulated by extracellular matrices.     

Interactions between extracellular matrix and growth factors in wound healing

Dynamic interactions between growth factors and extracellular matrix (ECM) are integral to wound healing. These interactions take several forms that may be categorized as direct or indirect. The ECM can directly bind to and release certain growth factors (e.g., heparan sulfate binding to fibroblast growth factor-2), which may serve to sequester and protect growth factors from degradation, and/or enhance their activity. Indirect interactions include binding of cells to ECM via integrins, which enables cells to respond to growth factors (e.g., integrin binding is necessary for vascular endothelial growth factor-induced angiogenesis) and can induce growth factor expression (adherence of monocytes to ECM stimulates synthesis of platelet-derived growth factor). Additionally, matrikines, or subcomponents of ECM molecules, can bind to cell surface receptors in the cytokine, chemokine, or growth factor families and stimulate cellular activities (e.g., tenascin-C and laminin bind to epidermal growth factor receptors, which enhances fibroblast migration). Growth factors such as transforming growth factor-β also regulate the ECM by increasing the production of ECM components or enhancing synthesis of matrix degrading enzymes. Thus, the interactions between growth factors and ECM are bidirectional. This review explores these interactions, discusses how they are altered in difficult to heal or chronic wounds, and briefly considers treatment implications.     

Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin

Background: Cell-mediated immunity is impaired in uraemia. The recognition and ensuing interactions of immune cells, such as CD4+ T lymphocytes, with adhesive glycoproteins of the extra-cellular matrix (ECM) are mediated by integrins of the {beta}1 subfamily. We have previously demonstrated that uraemic sera inhibit the proliferation and adhesion of normal CD+ T cells to ECM components. In the present study, the adhesive capacity of CD4+ T lymphocytes of dialyzed patients (both haemodialysis [HD] and continuous ambulatory peritoneal dialysis [CAPD] was evaluated. Methods: Adhesion of CD4+ T cells from dialysis patients to intact ECM and its immobilized moieties, fibronectin (FN) and laminin (LN) was measured following phorbol-12-myristate-13-acetate (PMA) stimulation. In addition, cell surface expression of {beta}1 integrins (VLA 4-6) was determined by FACScan analysis. Results: Compared to normal cells, CD4+ T cells of dialysis patients demonstrated a significantly reduced adhesion to ECM, FN and LN (27-28 vs 52-55% P <0.001). This decreased adhesive capacity was not normalized upon incubation of the cells with normal sera. Cell surface expression of {beta}1 integrins was not modified. The inhibition of cell adhesion was more pronounced in CAPD patients (23-24% vs 29-30% in HD, P <0.02). Serum albumin correlated directly with cell adhesion. Aged HD patients' T cells demonstrated increased adhesion to ECM and its ligands, whereas a reverse trend was demonstrated in the CAPD group. Conclusions: T cells of dialysis patients exhibit abnormal adhesive activity, which may be due to an acquired cellular defect induced by uraemic milieu. CAPD patients show a greater degree of adhesion impairment, possibly due to their lower concentrations of serum albumin.     

The relationship between myocardial extracellular matrix remodeling and ventricular function.

Elevations in myocardial stress initiate structural remodeling of the heart in an attempt to normalize the imposed stress. This remodeling consists of cardiomyocyte hypertrophy and changes in the amount of collagen, collagen phenotype and collagen cross-linking. Since fibrillar collagen is a relatively stiff material, a decrease in collagen can result in a more compliant ventricle while an increase in collagen or collagen cross-linking results in a stiffer ventricle. If continued elevations in wall stress exceed the ability of the heart to compensate, then the ventricular wall thickness is disproportionately reduced compared to chamber volume and diastolic and systolic dysfunction ensues. This review describes the structural organization of collagen within the myocardium, discusses its effect on ventricular function and considers whether therapy aimed at reducing fibrosis is efficacious in heart failure. The evidence indicates that chamber stiffness can clearly be affected by alterations in both collagen quantity and quality, with the effect of changes in collagen concentration being modified by the extent of collagen cross-linking. The limited evidence available regarding the effects of collagen on systolic function indicates that pharmacological attempts to reduce interstitial collagen have a negative impact. Accordingly, a shift in treatment strategies directed more specifically at affecting collagen cross-linking, rather than reducing the concentration of collagen, may be warranted in the prevention of the adverse impact of collagen alterations on myocardial remodeling.     

NG2蛋白多糖促进肾小球系膜细胞增殖和细胞外基质积聚

目的 探讨NG2蛋白多糖参与肾小球硬化过程的可能机制。 方法 构建针对NG2的特异性shRNA（Psilencer-NG2）和对照shRNA（Psilencer-NC）。将Psilencer-NG2、Psilencer-NC和全长型NG2真核表达载体pcDNA/NG2以及空质粒pcDNAⅠ分别转染到体外培养的大鼠系膜细胞（RMC）。实时定量PCR和Western印迹检测转染质粒对NG2 的干扰效率和过表达效率;MTT法观察各组细胞增殖情况;流式细胞仪检测细胞周期变化;实时定量PCR检测各组层粘连蛋白（laminin）的表达。 结果 转染pcDNA/NG2后的HBZY-1细胞NG2 mRNA和蛋白表达水平均明显增加（P < 0.05,P < 0.05）,而针对NG2的特异性shRNA干扰导致HBZY-1细胞NG2 mRNA和蛋白表达水平均显著下调（P < 0.01;P < 0.01）,分别表明达到过表达和沉默NG2的效应。过表达NG2后,RMC中laminin β1表达显著增加（1.25±0.04比 1.00±0.09,P < 0.05）;RMC增殖明显,处于S期的细胞数明显增加,而G0/G1期的细胞数减少。而沉默NG2后,laminin β1表达显著减少（0.81±0.02比1.00±0.08,P < 0.05）;RMC增殖缓慢,处于G0/G1期的细胞数明显增加,而S期的细胞数减少。 结论 NG2蛋白多糖可能通过促进系膜细胞增殖和细胞外基质积聚参与肾小球硬化的过程。     

Glycation of extracellular matrix proteins impairs migration of immune cells

The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes‐related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T‐cells. To achieve our purpose, we used in vitro AGE‐modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T‐cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin‐Alexa‐fluor 488‐labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE‐bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE‐modified matrix, but not with soluble AGEs like BSA‐AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs‐modified matrix protein inhibits cell migration and adhesion of Jurkat T‐cells.     

Interaction between adrenomedullin and angiotensin II in DNA synthesis and extracellular matrix accumulation in cultured rat kidney interstitial cells

Background. To investigate the role of adrenomedullin (AM) in the regulation of renal fibrosis, we assessed the effects of AM on angiotensin II (AT II)-induced cell proliferation and extracellular matrix (ECM) accumulation in cultured NRK 49F cells, a cell line derived from normal rat kidney fibroblasts. Methods. Northern blot analysis was performed, using cDNA probes against rat AM, human calcitonin-receptor-like receptor (CRLR), human receptor-activity-modifying protein 2 (RAMP 2), human collagen α-1 (I) (Col-I), human fibronectin (FN), and rat transforming growth factor (TGF)-β1. Polymerase chain reaction (PCR) analysis for CRLR was amplified for 35 cycles. Cell proliferation was determined by the measurement of [³H]thymidine incorporation. Results. We have shown that NRK 49F cells express AM and its receptor, which consists of a CRLR and RAMP 2. Rat AM significantly inhibited cell proliferation and mRNA expression of ECM in the absence and presence of AT II through an AM receptor, because calcitonin gene-related peptide (8-37) [CGRP (8-37)], an antagonist to AM receptor, completely reversed these inhibitory actions. The inhibitory effects appeared to be mediated by a marked increase in intracellular cAMP. While AT II enhanced ECM accumulation by a process depending upon autocrine TGF-β1 secretion in NRK 49F cells, AM suppressed the induction of TGF-β1. Conclusions. The inhibitory action of AM on ECM accumulation may be caused by the suppression of TGF-β1. AM may play an important role in the inhibition of renal interstitial fibrosis under pathological conditions, through acting in an autocrine and/or paracrine fashion. Received: May 2, 2001 / Accepted: November 27, 2001     

Optical coherence tomography grading correlates with MRI T2 mapping and extracellular matrix content

Optical coherence tomography (OCT) and T2 mapping are emerging clinical imaging technologies with potential to detect subsurface changes in cartilage retaining a macroscopically intact articular surface. This study tests the hypothesis that OCT correlates with magnetic resonance imaging (MRI) T2 values, and that OCT signal is sensitive to cartilage matrix degeneration. Forty‐five osteochondral cores were harvested from five human tibial plateau explants after MRI T2 mapping. Cores underwent OCT imaging and were graded as follows: A, obvious birefringence; B, no birefringence; C, subsurface voids and/or irregular surface. Extracellular matrix content was determined and cores underwent histologic and polarized light microscopy (PLM) evaluation. Grade B and C cores had 25% higher superficial T2 values (p = 0.047) and 50% higher deep T2 values (p = 0.012) than grade A cores. Grade B and C cores had 36% higher glycosaminoglycan (GAG) content compared to grade A cores (p = 0.009). Histology and PLM demonstrated increased surface irregularity and structural disorganization with increasing OCT grade. OCT grade and T2 value increased with increasing collagen disorganization, suggesting that MRI T2 mapping and OCT are sensitive to changes in collagen structure. Our results demonstrate the ability of OCT and T2 mapping to detect early cartilage degeneration in clinically normal appearing cartilage. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:546–552, 2010