Extracellular matrix formation by epithelial cells from human polycystic kidney cysts in culture.

Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [3H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [alpha (III)]3 and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M(r) of alpha 1(I) and alpha 2(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize alpha 1(I) and alpha 2(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cytogenetic mechanisms in vivo.     

Apoptosis in polycystic kidney disease

Apoptosis is the process of programmed cell death. It is a ubiquitous, controlled process consuming cellular energy and designed to avoid cytokine release despite activation of local immune cells, which clear the cell fragments. The process occurs during organ development and in maintenance of homeostasis. Abnormalities in any step of the apoptotic process are associated with autoimmune diseases and malignancies. Polycystic kidney disease (PKD) is the most common inherited kidney disease leading to end-stage renal disease (ESRD). Cyst formation requires multiple mechanisms and apoptosis is considered one of them. Abnormalities in apoptotic processes have been described in various murine and rodent models of PKD as well as in human PKD kidneys. The purpose of this review is to outline the role of apoptosis in progression of PKD as well as to describe the mechanisms involved. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

Epithelin mRNA expression in polycystic kidney disease

Epithelins are polypeptides that are preferentially expressed in epithelial cells and modulate growth. Epithelin expression is predominant in tissues of epithelial origin such as the kidney, spleen, lung, placenta, and colon. Because polycystic kidney disease involves abnormal proliferation of the proximal and/or distal tubule epithelial cells, we investigated epithelin mRNA expression in polycystic kidneys of mice homozygous for the mutation. Epithelin mRNA was highly expressed in the polycystic kidneys of homozygous mice when compared with the heterozygotes or wild type controls. A study on the time course of epithelin expression indicated that epithelin mRNA expression paralleled cyst formation and progression of the disease. A 2-fold increase in expression was observed at Day 15, a stage when cystic changes were first visible. This increase in expression was also observed at Day 21, a stage of maximum disease pathology, which ultimately results in the death of the animal. In situ hybridization localized epithelin mRNA predominantly to the epithelial cell layer surrounding the cysts. The high levels of epithelin in epithelial cells suggest a role in renal epithelial cell proliferation and cyst formation in polycystic kidney disease.     

Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oysterPinctada fucata

Summary To identify the cells which produce the extracellular matrix during bivalve wound healing, we observed epithelial regeneration inPinctada fucata and evaluated the ability of amebocytes to produce the matrix in vitro. Between days 1 and 3 after an ovary was implanted with abiotic material (a shell ball) via an incision, agranular amebocytes formed a sheath, consisting of 10–20 cell layers, between the implant and incised ovarian tissue. Extracellular matrix was deposited in the spaces between the amebocytes in the sheath. At the incised follicle, gonadal epithelial cells were attached to the newly formed matrix. When a mantle allograft (2 mm square) was implanted with abiotic material to bring them into close contact, epithelial cells emigrated from the allograft along the surface of the abiotic material where they attached to the newly formed matrix at the sheath of amebocytes. In vitro, agranular amebocytes formed a matrix composed of fibrils with a diameter of 20 nm during a 6-day culture period. Pepsin-digested extract of the cell layer forming the matrix gave protein bands with electrophoretic mobilities identical to - and -sized components of a collagen purified from this animal. The matrix exhibited immunoreaction to antiserum raised against the collagen and was stained by alcian bluc. Thus, the agranular amebocyte apparently has the ability to produce an extracellular matrix containing collagen and possibly proteoglycan(s).     

人脐带间充质干细胞微囊减轻小鼠急性肾损伤的研究

目的探讨人脐带间充质干细胞（MSCs）源性细胞外囊泡Oct-4 mRNA对受损的肾小管上皮细胞修复的作用及相关机制。 方法将培养的缺氧损伤肾小管上皮细胞置于含有人脐带MSCs细胞外囊泡及不同对照培养液的培养腔室玻片上孵育48?h,应用BrdU及TUNEL染色,检测各组细胞增殖或凋亡情况。将急性肾损伤模型小鼠分为4组：空白组、EVs组、Oct-4过表达组、Oct-4低敲组。并按照分组分别注射磷酸盐缓冲液（Vehicle）,人脐带MSCs细胞外囊泡（EVs）,过表达Oct-4基因的人脐带MSCs细胞外囊泡（EVs?+?Oct-4）及敲除Oct-4基因的人脐带MSCs外囊泡（EVs-Oct-4）,并在注射48?h及2周后采血测肌酐（Crea）及尿素氮（BUN）,了解肾功能变化;对各组上述处理后的肾组织应用TUNEL与增殖细胞核抗原表达量检测各组肾脏细胞凋亡与增殖情况;通过Masson染色检测了各组肾脏纤维化程度;通过PCR探索肾损伤后肾组织细胞Snail基因的表达变化。数据分析采用方差分析和SNK-q检验。 结果EVs?+ Oct-4处理缺氧的肾小管上皮细胞48?h后,TUNEL染色显示具有最少的凋亡细胞数（0~1）/?HPF,BrdU显示有最多的增殖细胞（7±2）/HPF。EVs,EV-Oct-4以及Vehicle对体外缺氧肾小管上皮细胞的上述作用依次减弱（P?相似文献   

Extracellular matrix dynamics during vertebrate axis formation

The first evidence for the dynamics of in vivo extracellular matrix (ECM) pattern formation during embryogenesis is presented below. Fibrillin 2 filaments were tracked for 12 h throughout the avian intraembryonic mesoderm using automated light microscopy and algorithms of our design. The data show that these ECM filaments have a reproducible morphogenic destiny that is characterized by directed transport. Fibrillin 2 particles initially deposited in the segmental plate mesoderm are translocated along an unexpected trajectory where they eventually polymerize into an intricate scaffold of cables parallel to the anterior-posterior axis. The cables coalesce near the midline before the appearance of the next-formed somite. Moreover, the ECM filaments define global tissue movements with high precision because the filaments act as passive motion tracers. Quantification of individual and collective filament "behaviors" establish fate maps, trajectories, and velocities. These data reveal a caudally propagating traveling wave pattern in the morphogenetic movements of early axis formation. We conjecture that within vertebrate embryos, long-range mechanical tension fields are coupled to both large-scale patterning and local organization of the ECM. Thus, physical forces or stress fields are essential requirements for executing an emergent developmental pattern-in this case, paraxial fibrillin cable assembly.     

Polycystins, focal adhesions and extracellular matrix interactions

Polycystic kidney disease is the most common heritable disease in humans. In addition to epithelial cysts in the kidney, liver and pancreas, patients with autosomal dominant polycystic kidney disease (ADPKD) also suffer from abdominal hernia, intracranial aneurysm, gastrointestinal cysts, and cardiac valvular defects, conditions often associated with altered extracellular matrix production or integrity. Despite more than a decade of work on the principal ADPKD genes, PKD1 and PKD2, questions remain about the basis of cystic disease and the role of extracellular matrix in ADPKD pathology. This review explores the links between polycystins, focal adhesions, and extracellular matrix gene expression. These relationships suggest roles for polycystins in cell-matrix mechanosensory signaling that control matrix production and morphogenesis. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Extracellular matrix fibrils and cell contacts in the chick embryo

Summary The migration of neural crest and sclerotome cells and the extension of ventral root axons in chick embryos at stages 16–20 were studied by light microscopy as well as scanning and transmission electron microscopy at the leg bud level of fixed specimens. Extensive cellular movements take place in association with an extracellular matrix consisting of microfibrils. The neural crest and sclerotome cells migrate into the large matrix-filled extracellular space surrounding the neural tube and notochord, apparently using microfibril bundles as substratum. The cells exhibit pseudopodia which are closely associated with the matrix fibrils. The fibrils around the notochord show a spatial arrangement indicating that the sclerotome cells are contact-guided to their subsequent positions. Mutual cell contacts, including those established by cell processes, frequently show cytoplasmic electron dense plaques at adjacent membranes. These small plaque contacts might be correlated to contact inhibition of locomotion between the cells and participate in the guidance of cells. The growth cones of extending axons exhibit filopodia contacting both surrounding mesenchyme cells and extracellular fibrils. The orientation of the axons might thus be affected by contacts with cell surfaces as well as with extracellular material.Technical assistance was given by Mrs. Kerstin Ahlfors, Mrs. Charlotte Fällström, Mrs. Annika Kylberg and Mrs. Stine SöderströmSupported by grants from The Swedish Natural Science Research Council     

Appearance and endocytic activity of epithelial cells from human efferent ducts in primary monolayer culture

Summary The culture of epithelial cells lining human efferent ducts, obtained from prostatic carcinoma patients, is described. Ciliated cells were observed to beat for at least one month on plastic. On pervious filters low cuboidal cells characterized the monolayers. Cells comprising monolayers over the filter were 5 to 9 m in height whereas taller cells were found over the original fragments (14 m). Some non-ciliated cells contained dark and light vacuoles, others were found to lack them. Both non-ciliated and ciliated cells maintained tight junctional complexes restricting the paracellular movement of horseradish peroxidase. Both types of cultured cells exhibited fluid-phase and adsorptive endocytosis from both apical and basal surfaces. It is reported for the first time that the monolayers form high resistance barriers (150 cm²) that prevent the apical medium from draining to the basal compartment over 24 h.     

Mouse models of polycystic kidney disease induced by defects of ciliary proteins

Polycystic kidney disease (PKD) is a common hereditary disorder which is characterized by fluid-filled cysts in the kidney. Mutation in either PKD1, encoding polycystin-1 (PC1), or PKD2, encoding polycystin-2 (PC2), are causative genes of PKD. Recent studies indicate that renal cilia, known as mechanosensors, detecting flow stimulation through renal tubules, have a critical function in maintaining homeostasis of renal epithelial cells. Because most proteins related to PKD are localized to renal cilia or have a function in ciliogenesis. PC1/PC2 heterodimer is localized to the cilia, playing a role in calcium channels. Also, disruptions of ciliary proteins, except for PC1 and PC2, could be involved in the induction of polycystic kidney disease. Based on these findings, various PKD mice models were produced to understand the roles of primary cilia defects in renal cyst formation. In this review, we will describe the general role of cilia in renal epithelial cells, and the relationship between ciliary defects and PKD. We also discuss mouse models of PKD related to ciliary defects based on recent studies. [BMB Reports 2013; 46(2): 73-79]     

Extracellular matrix formation by chondrocytes in monolayer culture

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.     

Extracellular matrix proteins regulate epithelial–mesenchymal transition in mammary epithelial cells

Mouse mammary epithelial cells undergo transdifferentiation via epithelial–mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.     

Extracellular matrix and integrin signaling in lens development and cataract

During development of the vertebrate lens there are dynamic interactions between the extracellular matrix (ECM) of the lens capsule and lens cells. Disruption of the ECM causes perturbation of lens development and cataract. Similarly, changes in cell signaling can result in abnormal ECM and cataract. Integrins are key mediators of ECM signals and recent studies have documented distinct repertoires of integrin expression during lens development, and in anterior subcapsular cataract (ASC) and posterior caspsule opacification (PCO). Increasingly, studies are being directed to investigating the signaling pathways that integrins modulate and have identified Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK) as downstream kinases that mediate proliferation, differentiation and morphological changes in the lens during development and cataract formation.     

Extracellular matrix networks in bone remodeling

Bones are constantly remodeled throughout life to maintain robust structure and function. Dysfunctional remodeling can result in pathological conditions such as osteoporosis (bone loss) or osteosclerosis (bone gain). Bone contains 100s of extracellular matrix (ECM) proteins and the ECM of the various bone tissue compartments plays essential roles directing the remodeling of bone through the coupled activity of osteoclasts (which resorb bone) and osteoblasts (which produce new bone). One important role for the ECM is to serve as a scaffold upon which mineral is deposited. This scaffold is primarily type I collagen, but other ECM components are involved in binding of mineral components. In addition to providing a mineral scaffolding role, the ECM components provide structural flexibility for a tissue that would otherwise be overly rigid. Although primarily secreted by osteoblast-lineage cells, the ECM regulates cells of both the osteoblast-lineage (such as progenitors, mature osteoblasts, and osteocytes) and osteoclast-lineage (including precursors and mature osteoclasts), and it also influences the cross-talk that occurs between these two oppositional cells. ECM influences the differentiation process of mesenchymal stem cells to become osteoblasts by both direct cell-ECM interactions as well as by modulating growth factor activity. Similarly, the ECM can influence the development of osteoclasts from undifferentiated macrophage precursor cells, and influence osteoclast function through direct osteoclast cell binding to matrix components. This comprehensive review will focus on how networks of ECM proteins function to regulate osteoclast- and osteoblast-mediated bone remodeling. The clinical significance of these networks on normal bone and as they relate to pathologies of bone mass and geometry will be considered. A better understanding of the dynamic role of ECM networks in regulating tissue function and cell behavior is essential for the development of new treatment approaches for bone loss.     

Global cytoskeletal control of mechanotransduction in kidney epithelial cells

Studies of mechanotransduction mediated by stress-sensitive ion channels generally focus on the site of force application to the cell. Here we show that global, cell-wide changes in cytoskeletal structure and mechanics can regulate mechanotransduction previously shown to be triggered by activation of the mechanosensitive calcium channel, polycystin-2, in the apical primary cilium of renal epithelial cells [S.M. Nauli, F.J. Alenghat, Y. Luo, E. Williams, P. Vassilev, X. Li, A.E. Elia, W. Lu, E.M. Brown, S.J. Quinn, D.E. Ingber, J. Zhou, Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33 (2003) 129-37]. Disrupting cytoplasmic microfilaments or microtubules in these cells eliminated fluid shear stress-induced increase of intracellular calcium. Altering the cytoskeletal force balance by inhibiting actomyosin-based tension generation (using 2,3-butanedione monoxime), interfering with microtubule polymerization (using nocodazole, cochicine, or taxol), or disrupting basal integrin-dependent extracellular matrix adhesions (using soluble GRGDSP peptide or anti-beta1 integrin antibody), also inhibited the calcium spike in response to fluid stress. These data indicate that although fluid stress-induced displacement of the primary cilium may be transduced into a calcium spike through activation of polycystin-2 and associated calcium-induced calcium release from intracellular stores, this mechanotransduction response is governed by global mechanical cues, including isometric tension (prestress) within the entire cytoskeleton and intact adhesions to extracellular matrix.     

Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro

Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using pre-osteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, β-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a pre-maturational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern.