3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.     

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2)

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.     

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.     

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression.     

TGFβ signaling and the control of myofibroblast differentiation: Implications for chronic inflammatory disorders

The myofibroblast is a highly specialized cell type that plays a critical role during normal tissue wound healing, but also contributes pathologically to chronic inflammatory conditions such as fibrosis and cancer. As fibrotic conditions continue to be a major burden to the public health system, novel therapies that target the function of myofibroblasts may show promise in the clinic. The cytokine transforming growth factor β (TGFβ) is the most potent known inducer of myofibroblast differentiation and thus represents a powerful target to modify myofibroblast function during disease. This review focuses on our current understanding of the key signaling pathways activated by TGFβ during myofibroblast differentiation.     

Up-regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in articular chondrocytes is mediated by serine/threonine and tyrosine kinases

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-β), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-β-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-β induction of TIMP-3. H7 and genistein also suppressed TGF-β-induced TIMP-3 protein expression. These results suggest that TGF-β signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517–527, 1998. © 1998 Wiley-Liss, Inc.     

Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9

To elucidate the roles of fibroblast growth factors (FGF) in tooth development, we have analyzed the expression patterns of fibroblast growth factor receptors (FGFR) in mouse teeth by in situ hybridization and studied the effects of FGF-2, -4, -8, and -9 on cell proliferation in vitro by local application with beads on isolated dental mesenchymes. mRNAs of FGFR-1, -2, and -3 were localized by probes specific for the alternative splice variants IIIb and IIIc. The expression patterns of FGFR1, -2, and -3 were completely different, and the two splicing variants of FGFR1 and 2 exhibited different expression domains. FGFR4 was not expressed in the developing teeth. The IIIb splice forms of FGFR1 and -2 were expressed in the dental epithelium during morphogenesis. The IIIc splice form of FGFR1 was expressed both in epithelium and mesenchyme whereas FGFR2 IIIc was confined to the mesenchymal cells of the dental follicle. Both splice forms of FGFR3 were expressed in dental papilla mesenchyme. None of the FGF-receptors was detected in the primary enamel knot, the putative signaling center regulating tooth morphogenesis. This may explain the fact that enamel knot cells do not proliferate, although they express intensely mitogenic FGFs. Beads releasing FGF-2, -4, -8, or -9 proteins stimulated cell proliferation in cultured dental mesenchymes. These data, together with our earlier data on FGF expression [Kettunen and Thesleff (1998): Dev Dyn 211:256–268] suggest that FGF-8 and -9 mediate epithelial-mesenchymal interactions during tooth initiation. During advancing morphogenesis FGF-3, -4, and -9 may act both on mesenchyme and epithelium. Finally, the intense expression of FGFR1 in odontoblasts and ameloblasts, and FGFR2 IIIb in ameloblasts suggests that FGFs participate in regulation of their differentiation and/or secretory functions. Dev. Genet. 22:374–385, 1998. © 1998 Wiley-Liss, Inc.     

PGE2 induces apoptosis of hepatic stellate cells and attenuates liver fibrosis in mice by downregulating miR-23a-5p and miR-28a-5p

MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl₄ and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis.     

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.