Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.

Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 μM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Ghrelin gene-related peptides : multifunctional endocrine/autocrine modulators in health and disease

Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, it has direct effects on cancer cell proliferation. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their role in obesity, diabetes and cancer.

The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (≤180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding α5β1- and αv-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of α5β1- and αv-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.

Adult human articular chondrocytes in a microcarrier-based culture system : expansion and redifferentiation

xpanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer-expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications.

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p < 0.05) by a factor of 4-5 from the top 0.1 mm (28 +/- 13 kPa, 141 +/- 10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p < 0.001), and correlated with the modulus (both p < 0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues. (c) 2005 Elsevier Ltd. All rights reserved.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

NMR measurement of 39K detectability and relaxation constants in rat tissue

Differences in the NMR detectability of 39K in various excised rat tissues (liver, brain, kidney, muscle, and testes) have been observed. The lowest NMR detectability occurs for liver (61 ± 3% of potassium as measured by flame photometry) and highest for erythrocytes (100 ± 7%). These differences in detectability correlate with differences in the measured 39K NMR relaxation constants in the same tissues. 39K detectabilities were also found to correlate inversely with the mitochondrial content of the tissues. Mitochondria prepared from liver showed greatly reduced 39K NMR detectability when compared with the tissue from which it was derived, 31.6 ± 9% of potassium measured by flame photometry compared to 61 ± 3%. The detectability of potassium in mitochondria was too low to enable the measurement of relaxation constants. This study indicates that differences in tissue structure, particularly mitochondrial content are important in determining 39K detectability and measured relaxation rates.

Protein monoubiquitination and polyubiquitination generate structural diversity to control distinct biological processes

Ubiquitination involves the attachment of ubiquitin (Ub) to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Polyubiquitination through different lysines (seven) or the N-terminus of Ub can generate different protein-Ub structures. These include monoubiquitinated proteins, polyubiqutinated proteins with homotypic chains through a particular lysine on Ub or mixed polyubiquitin chains generated by polymerization through different Ub lysines. The ability of the ubiquitination pathway to generate different protein-Ub structures provides versatility of this pathway to target proteins to different fates. Protein ubiquitination is catalyzed by Ub-conjugating and Ub-ligase enzymes, with different combinations of these enzymes specifying the type of Ub modification on protein substrates. How Ub-conjugating and Ub-ligase enzymes generate this structural diversity is not clearly understood. In the current review, we discuss mechanisms utilized by the Ub-conjugating and Ub-ligase enzymes to generate structural diversity during protein ubiquitination, with a focus on recent mechanistic insights into protein monoubiquitination and polyubiquitination.