Search

The ability to predict and guide stem cell differentiation remains a major challenge in regenerative medicine. Numerous dynamic microenvironmental cues often provide synergistic or combinatorial signals that influence the fate of stem cells, and ultimately drive functional tissue formation. This interplay between microenvironmental cues within tissues is under intense investigation. Our goal was to better understand this interplay within the framework of a systematic 3D platform that would enable high-throughput screening (HTS) of factors that contribute to stem cell fate decisions. It is important that such platforms provide valid biomimetic microenvironments, which can be translated to macroscale constructs. Specifically, we reported on a technique for screening of combinatorial 3D niches to guide the osteogenic differentiation of human mesenchymal stem cells (hMSCs). This platform offers a rapid, cost-effective and multiplexed approach for a variety of tissue engineering applications. [ABSTRACT FROM PUBLISHER]

Abstract: Interleukin-11 (IL-11) is a multifunctional cytokine involved in various pathways in blood cells, their precursors and many other cell types in vitro and in vivo. The effects of IL-11 are largely mediated by the IL-11 receptor α-chain (IL-11Rα). In this study, a putative cDNA sequence encoding the 414 amino acid propeptide of chicken IL-11R (chIL-11R) was identified. The predicted 414 amino acid sequence showed 42–43% sequence identity with mammalian homologues. In a domain search of the molecule, two fibronectin (FN) type-III domains were identified in the C- terminal portion. On comparison with mammalian IL-11R, 4 conserved cysteine residues and a WSXWS motif were observed within the FN type-III domains. Expression analysis revealed that chIL-11Rα is strongly expressed in brain, heart, lung, liver, glandular stomach, kidney, the immature testis, ovary and chicken blastodermal cells (CBCs) after 1-day-cultivation. These findings strongly indicate that the identified chicken cDNA sequence encodes chIL-11R α-chain homologue. [Copyright &y& Elsevier]

The efficiency of small interfering RNA (siRNA)-induced gene knockdown is hampered by low transfection efficiency. We established a novel and simple double transfection method using specific siRNA duplexes targeted against human thrombin receptor PAR-1 in DU 145 prostate cancer cells. The initial siRNA transfection of cell suspensions followed by re-transfection of adherent cells on the following day resulted in undetectable PAR-1 mRNA and absent receptor protein. PAR-1 mRNA expression was silenced for up to five days. Functional studies showed that PAR-1 gene silencing in DU 145 cells abolished the modulating effects of thrombin on cell adhesion to the extracellular matrix proteins, fibronectin and laminin, thus demonstrating the essential role of PAR-1 in mediating thrombin effects on DU 145 cell adhesion. [Copyright &y& Elsevier]

Syndecan-1 was overexpressed in T47D, MCF-7, or Hs578t human breast carcinoma cell lines, mimicking overexpression observed in carcinomas in vivo. Overexpression of syndecan-1, or its ectodomain alone fused to a glycosylphosphatidylinositol anchor (GPI-mS1ED), promotes cell rounding in 2D culture. Deletions within the syndecan-1 ectodomain (S1ED) implicate an active site within the core protein between the glycosaminoglycan attachment region and the transmembrane domain. Polyclonal antibodies directed against the ectodomain, or treatment with the tyrosine kinase inhibitor genistein, block activity and revert GPI-mS1ED overexpressing cells to a normal morphology. Extracellular matrix (ECM)-dependent signaling appears to be targeted, as GPI-mS1ED cells attach and spread similarly to control cells in response to E-cadherin engagement, but fail to spread on integrin-dependent ligands. However, integrin-dependent cell attachment, and integrin activation and subsequent FAK phosphorylation are unaffected, suggesting that the syndecan regulates the integration of signaling following matrix adhesion. In 3D culture, where syndecan-1 may have a more critical role in cell behavior, the disrupted signaling leads to poorly cohesive, invasive colonies. Thus, altered matrix-dependent signaling due to increased levels of cell surface syndecan-1 may lead to epithelial cell invasion during early stages of tumorigenesis. [Copyright &y& Elsevier]

Recent advances in molecular force measurements have resulted in the quantification of the nanomechanical properties of single molecular bonds, and elucidated novel relationships between molecular architecture and biomolecular adhesion. The measured forces to rupture single intermolecular bonds revealed novel and unexpected ways that proteins respond to mechanical force. Measurement of the magnitude of interprotein forces and the distances over which they act further determined how protein architecture may contribute to both the stability and structural organization of adhesive junctions. [Copyright &y& Elsevier]

Many cell interactions depend on the assembly of cell protrusions; these include cell attachment and migration in the extracellular matrix, cell–cell communication, and the ability of cells to sense their local environment. Cell protrusions are extensions of the plasma membrane that are supported internally by actin-based structures that impart mechanical stiffness. Fascin is a small, globular actin-bundling protein that has emerging roles in diverse forms of cell protrusions and in cytoplasmic actin bundles. The fascin–actin interaction is under complex regulation from the extracellular matrix, peptide factors and other actin-binding proteins. Recent developments advance our understanding of the multifaceted regulation of fascin and the roles of fascin-containing structures in cell adhesion, motility and invasion in the life of vertebrate organisms. [Copyright &y& Elsevier]

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks recognition and repair. However, Ku is expressed also on the surface of different types of cells along with its intracellular pool within the nucleus and the cytoplasm. Participation of membrane-associated Ku in cell–cell interaction has been reported recently. Here, we describe a novel function of cell-surface Ku as an adhesion receptor for fibronectin (Fn). The role of Ku in cell adhesion was investigated by comparing the Ku80 deficient Chinese hamster ovary (CHO) cell line, xrs-6, with clones transfected stably with either the hamster or human Ku80 cDNA. Ku expression in transfectant cells resulted in a significant increased adhesion on Fn and type IV collagen as compared to control cells. The observed increase in cell adhesion relied on Ku cell-surface expression, since antibodies directed against Ku70 or Ku80 subunit inhibited adhesion on Fn of Ku80, but not control vector, transfected xrs-6 cells. In addition, both Ku70 and Ku80 present a structural relationship with integrin I (or A) domains and the A1 and A3 domains of von Willebrand factor, domains known to be involved in Fn binding. Both Ku70 and Ku80 exhibit a complete set of residues compatible in their position and chemical nature with the formation of a metal ion-dependent adhesion (MIDAS) site implicated in ligand binding and integrin activation. Taken together, these functional and structural approaches support a new role for Ku as an adhesion receptor for Fn. [Copyright &y& Elsevier]

Evidence has been emerging to suggest that integrin could induce growth inhibition in some cell types. Some of the molecular mechanisms underlying growth arrest have been elucidated. We reported here that overexpression of integrin β1 imposed a growth inhibitory effect on the hepatocellular carcinoma cell line SMMC-7721, and this phenomenon was mainly attributed to the cyclin-dependent kinase inhibitor p21CIP1. Furthermore, our findings suggested that transcription activity of the p21CIP1 gene could be upregulated in the integrin β1-overexpressing cells, and possibly controlled by the cis-elements in the core region of the p21CIP1 promoter. [Copyright &y& Elsevier]

Control of cell–matrix interactions is a central principle for the design of biomaterial in tissue engineering. In this study, we evaluated a synthetic glycopolymer, which is recognized by the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes, as an artificial matrix to regulate integrin-mediated signaling. The phosphorylation of focal adhesion kinase was restricted in hepatocytes cultured on the glycopolymer compared with fibronectin. In addition, there was no reorganization of cytoskeleton-related proteins such as actin filaments, microtubules, and vinculin in hepatocytes cultured on the glycopolymer. DNA synthesis and cyclin D1 expression were suppressed in hepatocytes grown on the glycopolymer as compared with those grown on fibronectin and collagen. The data suggest that the glycopolymer will be a good artificial matrix to regulate integrin-mediated signaling and cell growth through the unique ASGPR–carbohydrate interaction. [Copyright &y& Elsevier]

C-protein (MyBP-C) is a myosin-binding protein that is usually seen in two sets of seven to nine positions in the C-zones in each half of the vertebrate striated muscle A-band. Skeletal muscle C-protein is a modular structure containing ten sub-domains (C1 to C10) of which seven are immunoglobulin-type domains and three (C6, C7 and C9) are fibronectin-like domains. Cardiac muscle C-protein has an extra N-terminal domain (C0) and also some sequence insertions, one of which provides phosphorylation sites. It is conceivable that C-protein has both a structural and regulatory role within the sarcomere. The precise mode of binding of C-protein to the myosin filament has not been determined. However, detailed ultrastructural studies have suggested that C-protein, which binds to myosin, can give rise to a longer periodicity (about 435 A˚) than the intrinsic myosin filament repeat of 429 A˚. The reason for this has remained a puzzle for over 25 years. Here we show by modelling and computation that the presence of this longer periodicity could be explained if the myosin-binding part of C-protein binds to myosin with the expected 429 A˚ repeat, but if there are systematic interactions of the N-terminal end of C-protein with the neighbouring actin filaments in the hexagonal lattice of filaments in the A-band. We also show that if they occur these interactions would probably only arise in defined muscle states. Further analysis of the MyBP-C sequence identifies a possible actin-binding domain in the Pro-Ala-rich sequence found at the N terminus of skeletal MyBP-C and between domains C0 and C1 in the cardiac sequence. [Copyright &y& Elsevier]

The large multidomain muscle protein myosin binding protein C (MyBP-C) has been implicated for some time in cardiac disease while until recently little was known about its structure and function. Here we present a detailed study of the central domain C5 of the cardiac isoform of MyBP-C. This domain is unusual in several aspects. Firstly it contains two sizeable insertions compared to the non-cardiac isoforms. The first insertion comprises the linker between domains cC4 and cC5 that is elongated by ten amino acid residues, the second insertion comprises an elongation of the CD-loop in the middle of the domain by ∼30 amino acid residues. Secondly two point mutations linked to familial hypertrophic cardiomyopathy (FHC) have been identified in this domain. This work shows that the general fold of cC5 is in agreement with the IgI family of β-sandwich structures. The long cardiac-specific linker between cC4 and cC5 is not a linker at all but an integral part of the fold of cC5, as evidenced by an unfolded mutant in which this segment was removed. The second insertion is shown to be unstructured, highly dynamic and mostly extended according to NMR relaxation measurements and analytical ultracentrifugation. The loss of several key interactions conserved in the CD-loop of the IgI fold is assumed to be responsible for the low stability of cC5 compared to other IgI domains from titin and MyBP-C itself. The low thermodynamic stability of cC5 is most evident in one of the two FHC-linked mutations, N755K (Asn115 in this construct) which is mainly unfolded with a small proportion of a native-like folded species. In contrast, the second FHC-linked mutation, R654H (Arg14 in this construct) is as well folded and stable as the wild-type. This residue is located in the extended β-bulge at the N terminus of the protein, pointing towards the surface of the CFGA′ β-sheet. This position is in agreement with recent data pointing to a function of Arg654 in an intermolecular interaction with MyBP-C domain cC8. [Copyright &y& Elsevier]

We observed in vitro that after treatment with the Arg-Gly-Asp (RGD) peptide, non-spreading Crassostrea gigas hemocytes underwent cell death. Utilizing a combination of a Hoechst staining method and a DNA fragmentation assay, the typical features of apoptosis were shown, i.e. cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. The hemocyte cell death caused by the RGD peptide appears to be sequence-specific, since no induction was shown in the alanine-substituted control peptide (RAD) treatment. Interestingly, the glutamic acid-substituted control peptide (RGE) also induced hemocytic cell death, but a different type of the death to that induced by the RGD peptide. This is the first report that specific peptides induce cell death in molluscan hemocytes. [Copyright &y& Elsevier]

Syndecans, a family of transmembrane proteoglycans, interact with numerous extracellular ligands through specific sequences in their heparan sulfate chains and have been considered to be co-receptors for matrix molecules and growth factors. In addition to their roles as co-receptors, many studies have recently suggested that signaling through core protein of syndecans can regulate cytoskeletal organization through their clustering, association with cytoskeletal structures, binding to cytoplasmic binding proteins, and intracellular phosphorylation. Here we will review current understanding of signaling through syndecans in cytoskeletal organization. [Copyright &y& Elsevier]

Transforming growth factor-β1 (TGF-β1) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-β1 in cultures of human periodontal ligament cells (HPL cells). TGF-β1 increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-β1 also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-β1. HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-α5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-β1. This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-β1 in HPL cells. [Copyright &y& Elsevier]

Experimental studies have indicated that FN-III modules undergo reversible unfolding as a mechanism of elasticity. The unfolding of FN-III modules, including the cell-binding FN-III10 module, has further been suggested to be functionally relevant by exposing buried cryptic sites or modulating cell binding. While steered molecular dynamics (SMD) simulations have provided one tool to investigate this process, computational requirements so far have limited detailed analysis to the early stages of unfolding. Here, we use an extended periodic box to probe the unfolding of FN-III10 for extensions longer than 60 A˚. Up to three plateaus, corresponding to three metastable intermediates, were observed in the extension-time profile from SMD stretching of FN-III10. The first and second plateaus correspond to a twisted and an aligned state prior to unraveling FN-III10 β-strands. The third plateau, at an extension of ∼100 A˚, follows unraveling of FN-III10 A and B-strands and precedes breaking of inter-strand hydrogen bonds between F and G-strands. The simulations revealed three forced unfolding pathways of FN-III10, one of which is preferentially selected under physiological conditions. Implications for fibronectin fibrillogenesis are discussed. [Copyright &y& Elsevier]

Trichomonosis is the vaginitis caused by Trichomonas vaginalis. This sexually transmitted agent achieves successful host parasitism through various means including: (1) acquisition of nutrients through specific receptors; (2) recognition and binding to mucin followed by cytoadherence mediated by adhesins that resemble metabolic enzymes; (3) evasion of immune responses through (i) masking of organisms by host proteins, (ii) shedding of trichomonad proteins into the secretions and (iii) secretions of cysteine proteinases that degrade all immunoglobulin subclasses and complement; (4) alternating surface expression of at least two antigen repertoires; and (5) alternate and coordinate expression of virulence genes in response to host environmental factors. The fact that the parasite survives long term in the varying and adverse environment of the vagina attests to the highly evolved nature of this protist. An understanding of the non-self-limiting nature of this infection may come from recent findings illustrating the complexity of Trichomonas vaginalis–fibronectin (FN) interactions. The parasite readily attaches to surfaces with immobilized FN and binds to FN in a highly specific receptor-mediated fashion. The amount and affinity of bound FN by live organisms is influenced by concentrations in medium of both iron and calcium. De novo protein synthesis is required for optimal FN acquisition in the presence of calcium. Furthermore, the parasites bind with differing affinities to the N-terminal domain (NTD), the cell-binding domain (CBD) and the gelatin-binding domain (GBD) of FN. Iron modulates binding of NTD similar to that of FN. This minireview summarizes recent findings on the T. vaginalis–FN associations. [Copyright &y& Elsevier]

The region of fibronectin (FN) surrounding the two type II modules of FN binds type I collagen. However, little is known about interactions of this collagen binding domain with other collagen types or extracellular matrix molecules. Among several expressed recombinant (r) human FN fragments from the collagen binding region of FN, only rI6–I7, which included the two type II modules and both flanking type I modules, bound any of several tested collagens. The rI6–I7 interacted specifically with both native and denatured forms of types I and III collagen as well as denatured types II, IV, V and X collagen with apparent Kd values of 0.2–3.7×10−7 M. Reduction with DTT disrupted the binding to gelatin verifying the functional requirement for intact disulfide bonds. The FN fragments showed a weak, but not physiologically important, binding to heparin, and did not bind elastin or laminin. The broad, but selective range of ligand interactions by rI6–I7 mirrored our prior observations for the collagen binding domain (rCBD) from matrix metalloproteinase-2 (MMP-2) [J. Biol. Chem. 270 (1995) 11555]. Subsequent experiments showed competition between rI6–I7 and rCBD for binding to gelatin indicating that their binding sites on this extracellular matrix molecule are identical or closely positioned. Two collagen binding domain fragments supported cell attachment by a β1-integrin-dependent mechanism although neither protein contains an Arg–Gly–Asp recognition sequence. Furthermore, activation of MMP-2 and MMP-9 was greatly reduced for HT1080 fibrosarcoma cells cultured on either of the fibronectin fragments compared to full-length FN. These observations imply that the biological activities of FN in the extracellular matrix may involve interactions with a broad range of collagen types, and that exposure to pathologically-generated FN fragments may substantially alter cell behavior and regulation. [Copyright &y& Elsevier]

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly. [Copyright &y& Elsevier]

In primary systemic vasculitis anti endothelial cell autoantibodies (AECA) have been described frequently. They represent a heterogeneous group of autoantibodies whose target antigens are mostly unknown. We tried to find AECA-antigens by a co-operative binding assay with a panel of monoclonal antibodies (mAb) directed to human umbilical vein endothelial cells (HUVEC) and extracellular matrix proteins. The mAb were used to bind antigens from lysate of endothelial cells, and binding of human antibodies to these antigens was measured. mAb directed to Vitronectin (VN) and Fibronectin (FN) resulted in enhanced binding of antibodies in sera from patients with Churg Strauss Syndrome (CSS) and Wegener''s Granulomatosis (WG) compared to normal sera. Neither free autoantibodies against VN or FN could be detected nor did the addition of endothelial cell lysate influence the binding activity from the patients'' sera. This suggests that preformed VN and FN-containing immune complexes (IC) are present in the patient sera. The amount of IC was decreased by incubation with HUVEC, demonstrating that these IC can bind to endothelial cells. However, their involvement in the pathogenesis of the disease is not clearly defined. Our data suggest that there are preformed IC present in sera of patients with CSS and WG that contain VN and FN and bind to endothelial cells. [Copyright &y& Elsevier]

Hepatocytes are the source of plasma fibronectin (FN) which lacks the alternatively spliced EDI segment, distinctive of oncofetal FN. When hepatic or other epithelial cells are cultured on plastic, EDI inclusion is triggered. Here we report that EDI inclusion is inhibited when hepatic cells are cultured on a basement membrane-like extracellular matrix (ECM), demonstrating a new role for the ECM in the control of gene expression. The effect is duplicated by collagen IV and laminin but not by collagen I; is not observed with another alternatively spliced FN exon (EDII); and correlates with a decrease in cell proliferation, consistently with high EDI inclusion levels observed in many physiological and pathological proliferative processes. [Copyright &y& Elsevier]

It is well established that mechanical forces can regulate cell growth and guide tissue remodeling, yet little is known about how mechanical signals act at the cell surface membrane to produce biochemical changes in the cell. To explore this question, I used a mouse embryonic F9 vinculin-deficient cell line (γ229), which, unlike wild-type cells, shows no fibronectin-dependent cell spreading. The wild-type cell line exhibited a twofold increase in area over four hours. I observed (i) an earlier rise in intracellular free calcium from ∼0.2 to ∼3 μm in wild-type compared with γ229 cells, thus similar calcium levels after 4 h; (ii) an initial higher ratio of p-MAP/MAP-Kinase for γ229, but similar FA-Kinase activation; and (iii) a marginal change in intracellular pH [pH]i in both F9 cell lines. When I applied controlled local stresses directly to integrin receptors using RGD-coated magnetic beads, they displaced to a lesser extent in wild-type than in γ229 cells. Both F9 cell lines showed a small stress-dependent rise in [Ca2+]i levels and similar PKA-c activity. In summary, the mechanical linkage of integrin-vinculin-cytoskeleton seemed not to be essential for chemical signal transduction. [Copyright &y& Elsevier]

Objectives: Angiotensin II (ANG II) can modulate cellular proliferation in various cell types via AT1 and AT2 receptors. In the present study, we investigated the effect of the angiotensin AT1 and AT2 receptors on DNA-synthesis as well as on the expression of the extracellular matrix (ECM) components, thrombospondin-1 (TSP-1) and fibronectin (FN) in endothelial cells (EC). Methods: The experiments were performed in microvascular EC derived from rat heart (CEC) and macrovascular EC derived from bovine aorta (BAEC). The experiments were performed in cells of the second and third passage and the expression of AT1 and AT2 receptors was verified by binding studies, Northern analysis or RT–PCR. Quiescent rat CEC and BAEC were stimulated to proliferate by the addition of 25 ng/ml bFGF, while ANG II (10−7 M) and the selective ANG II receptor antagonists, Losartan (10−5 M) and PD123177 (10−6 M) or the AT2 agonist, CGP42112A (10−7 M) were added 16 h later. Results: ANG II induced a dose-dependent decrease of DNA-synthesis in BAEC measured by [3H]-thymidine incorporation. This inhibitory effect of ANG II was prevented by the addition of the AT2 receptor antagonist PD123177 (10–6 M), demonstrating, that the inhibition of DNA synthesis is mediated by the AT2 receptor. In the presence of Losartan, stimulation of both, CEC and BAEC, with ANG II resulted in a marked increase of TSP-1 mRNA levels, which was maximal between 3 and 6 h in rat CEC and after 9 h in BAEC. In addition, TSP-1 was clearly induced by the AT2 agonist CGP42112A. In contrast, blockade of the AT2 receptor by the selective AT2 antagonist, PD123177 (10−6 M), resulted in a pronounced down regulation of FN mRNA 9 h after the stimulation. Conclusions: The present results suggest that the ANG II receptor subtype AT2 mediates growth inhibition in macrovascular EC similar to what has been shown before in microvascular rat EC and that AT2 receptors mediates remodeling of the endothelial ECM by upregulation of TSP-1 expression in both macro- and micro-vascular endothelial cells. [ABSTRACT FROM PUBLISHER]

Objectives: Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFβ-1 and osteopontin are involved in this process. Methods and results I: In a first set of experiments, the direct effect of Ang II on collagen I, TGFβ-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFβ-1 (183±21% of control, p<0.05) and osteopontin mRNA (275±58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95±8%). Stimulation with TGFβ-1 led to an increase in collagen I and III mRNA (127±10%, p<0.05; 140±15%, p<0.02). Methods and results II: In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFβ-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, III or fibronectin mRNA content after 24 h. In contrast, TGFβ-1 stimulation significantly increased collagen I and III mRNA expression (124±5% and 128±5%, p<0.002). Conclusions: In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFβ-1 and osteopontin mRNA expression. Since TGFβ-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart. [ABSTRACT FROM PUBLISHER]

Focal adhesions (FAs) provide the cells linkages to extracellular matrix (ECM) at sites of integrins binding and transmit mechanical forces between the ECM and the actin cytoskeleton. Cells sense and respond to physical stimuli from their surrounding environment through the activation of mechanosensitive signaling pathways, a process called mechanotransduction. In this study, we used RGD-peptide conjugated DNA tension gauge tethers (TGTs) with different tension tolerance (T tol ) to determine the molecular forces required for FA maturation in different sizes and YAP nuclear translocation. We found that the limitation of FA sizes in cells seeded on TGTs with different T tol were less than 1 μm, 2 μm, 3 μm, and 6 μm for T tol values of 43 pN, 50 pN, 54 pN, and 56 pN, respectively. This suggests that the molecular tension across integrins increases gradually as FA size increases throughout FA maturation. For YAP nuclear translocation, significant YAP nuclear localization was observed only in the cells seeded on the TGTs with T tol ≥ 54 pN, but not on TGTs with T tol ≤ 50 pN, suggesting a threshold of molecular force across integrins for YAP nuclear translocation lies in the range of 50 pN-54 pN., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Excessive fibrosis affects more than 100 million patients yearly, leading to the accumulation of extracellular matrix that compromises tissue architecture and impedes its function. Intrinsic properties of the amniotic membrane have alluded to its potential to inhibit excessive fibrosis; therefore, this study aimed to investigate the effects of dehydrated human amnion/chorion membrane (dHACM) on dermal fibroblasts and their role in fibrotic pathways. Human dermal fibroblasts were stimulated with TGFβ1, triggering myofibroblast-like characteristics in vitro. Subsequent addition of dHACM in the continued presence of TGFβ1 inhibited downstream signaling, leading to a reduction in the expression of known fibrotic and extracellular matrix genes. In addition, dHACM decreased alpha-smooth muscle actin, a stress filament responsible for contractile activity in scarring. The functional outcome of these effects was observed in an ex vivo model for cellular contraction. Hyperactivation of TGFβ signaling increased the contractile capacity of myofibroblasts embedded within a collagen substrate. Simultaneous addition of dHACM treatment prevented the marked contraction, which is likely a direct result of the inhibition of TGFβ signaling mentioned earlier. These observations may support the use of dHACM in the regulation of fibroblast activity as it relates to tissue fibrosis., (© 2021 The Authors.)

This study analyzed the expression of extracellular matrix (ECM) proteins during aortic valve calcification with mass spectrometry, and further validated in an independent human cohort using RNAseq data. The study reveals that valve calcification is associated with significant disruption in ECM and metabolic pathways, and highlights a strong connection between metabolic markers and ECM remodeling. It also identifies FNDC1 and MXRA5 as novel ECM biomarkers in calcified valves, electing them as potential targets in the development and progression of aortic stenosis., Competing Interests: Dr. Bouchareb was supported in part by a National Institutes of Health (NIH) grant T32 HL7824-19; Dr. Lebeche was supported by National Institutes of Health grant R01HL137220. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2021 The Authors.)

Current pre-clinical evidences of Centella focus on its pharmacological effects on normal wound healing but there are limited studies on the bioactivity of Centella in cellular dysfunction associated with diabetic wounds. Hence we planned to examine the potential of Centella cordifolia in inhibiting methylglyoxal (MGO)-induced extracellular matrix (ECM) glycation and promoting the related cellular functions. A Cell-ECM adhesion assay examined the ECM glycation induced by MGO. Different cell types that contribute to the healing process (fibroblasts, keratinocytes and endothelial cells) were evaluated for their ability to adhere to the glycated ECM. Methanolic extract of Centella species was prepared and partitioned to yield different solvent fractions which were further analysed by high performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) method. Based on the antioxidant [2,2-diphenyl-1-picrylhydrazyl (DPPH) assay] screening, anti-glycation activity and total phenolic content (TPC) of the different Centella species and fractions, the ethyl acetate fraction of C. cordifolia was selected for further investigating its ability to inhibit MGO-induced ECM glycation and promote cellular distribution and adhesion. Out of the three Centella species ( C. asiatica, C. cordifolia and C. erecta ), the methanolic extract of C. cordifolia showed maximum inhibition of Advanced glycation end products (AGE) fluorescence (20.20 ± 4.69 %, 25.00 ± 3.58 % and 16.18 ± 1.40 %, respectively). Its ethyl acetate fraction was enriched with phenolic compounds (3.91 ± 0.12 mg CAE/μg fraction) and showed strong antioxidant (59.95 ± 7.18 μM TE/μg fraction) and antiglycation activities. Improvement of cells spreading and adhesion of endothelial cells, fibroblasts and keratinocytes was observed for ethyl acetate treated MGO-glycated extracellular matrix. Significant reduction in attachment capacity of EA.hy926 cells seeded on MGO-glycated fibronectin (41.2%) and attachment reduction of NIH3t3 and HaCaT cells seeded on MGO-glycated collagen (33.7% and 24.1%, respectively) were observed. Our findings demonstrate that ethyl acetate fraction of C. cordifolia was effective in attenuating MGO-induced glycation and cellular dysfunction in the in-vitro wound healing models suggesting that C. cordifolia could be a potential candidate for diabetic wound healing. It could be subjected for further isolation of new phytoconstituents having potential diabetic wound healing properties., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)