Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

A connection between extracellular matrix and hormonal signals during the development of the human fetal adrenal gland

The human adrenal cortex, involved in adaptive responses to stress, body homeostasis and secondary sexual characters, emerges from a tightly regulated development of a zone-specific secretion pattern during fetal life. Its development during fetal life is critical for the well being of pregnancy, the initiation of delivery, and even for an adequate adaptation to extra-uterine life. As early as from the sixth week of pregnancy, the fetal adrenal gland is characterized by a highly proliferative zone at the periphery, a concentric migration accompanied by cell differentiation (cortisol secretion) and apoptosis in the central androgen-secreting fetal zone. After birth, a strong reorganization occurs in the adrenal gland so that it better fulfills the newborn's needs, with aldosterone production in the external zona glomerulosa, cortisol secretion in the zona fasciculata and androgens in the central zona reticularis. In addition to the major hormonal stimuli provided by angiotensin II and adrenocorticotropin, we have tested for some years the hypotheses that such plasticity may be under the control of the extracellular matrix. A growing number of data have been harvested during the last years, in particular about extracellular matrix expression and its putative role in the development of the human adrenal cortex. Laminin, collagen and fibronectin have been shown to play important roles not only in the plasticity of the adrenal cortex, but also in cell responsiveness to hormones, thus clarifying some of the unexplained observations that used to feed controversies.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Nucleotides as Antiviral Compounds. On the Feasibility of an Esterase-dependent Prodrug Strategy for 2-5A

Nukleotidien ja oligonukleotidien analogeilla on merkittävä rooli virusten aiheuttamien tautien hoidossa. Tämän kaltaiset yhdisteet voivat estää spesifisesti virusten proteiineja tai aktivoida luontaista immuunijärjestelmää, jossa 2-5A:ksi kutsutut lyhyet 2´,5´-sitoutuneet oligomeerit ovat keskeisiä tekijöitä. Nukleotideihin ja oligonukleotideihin pohjautuvien lääkkeiden tehokkuus riippuu pääasiassa aihiolääkestrategiasta, jolla niiden sisäänottoa soluun tehostetaan. Tavanomaisessa aihiolääkestrategiassa negatiivisesti varautuneet fosfaattiryhmät suojataan rasvaliukoisilla biohajoavilla suojaryhmillä, jotta molekyyli läpäisee solukalvon helpommin. Solun sisällä aihiolääke muuttuu aktiiviseksi lääkeaineeksi, kun suojaryhmät irtoavat solun entsyymien, kuten esteraasien vaikutuksesta. Väitöskirjassa arvioitiin esteraasin katalysoiman aihiolääkestrategian soveltuvuutta 2-5A-trimeerille syntetisoimalla kaksi erilaista 2-5A-aihiolääkekandidaattia ja tutkimalla 2-5A:n purkautumista karboksiesteraasi-entsyymin vaikutuksesta. Suojaryhmäsuunnitelma perustui esteraasilabiileihin 2,2-disubstituoituihin asyylioksipropyyliryhmiin ja asyylioksimetyyliryhmiin, joilla suojattiin trimeerien fosfaatti- ja 3´-hydroksyyliryhmät. Tulokset osoittivat, että esteraasilabiilien suojaryhmien irtoaminen 2-5A:sta hidastui merkittävästi, kun yhdisteeseen kertyi negatiivista varausta. Lisäksi suojaryhmien hajotessa muodostui elektrofiilisiä alkyloivia aineita, jotka ovat mahdollisesti toksisia. Näistä syistä johtuen kehitettiin kuusi uudenlaista 2,2,-disubstituoitua 4-asyylitio- 3-oksobutyyliryhmää fosfodiestereiden suojaamiseksi. Suojaryhmät irtoavat sekä esteraasin katalysoimana, että lämpötilan vaikutuksesta. Tämä on hyödyllinen ominaisuus silloin, kun entsyymin affiniteetti negatiivisesti varattuun substraattiin heikkenee. Suojaryhmien hydrolyyttinen ja entsymaattinen stabiilisuus on helposti säädeltävissä, jotta suojauksen purkautumisen nopeus voidaan optimoida. Vapautuneet suojaryhmät eivät ole merkittävästi alkyloivia, sillä niiden ei havaittu alkyloivan glutationia.

Effect of photodynamic therapy on the extracellular matrix and associated components

In many countries, photodynamic therapy (PDT) has been recognized as a standard treatment for malignant conditions (for example, esophageal and lung cancers) and non-malignant ones such as age-related macular degeneration and actinic keratoses. The administration of a non-toxic photosensitizer, its selective retention in highly proliferating cells and the later activation of this molecule by light to form reactive oxygen species that cause cell death is the principle of PDT. Three important mechanisms are responsible for the PDT effectiveness: a) direct tumor cell kill; b) damage of the tumor vasculature; c) post-treatment immunological response associated with the leukocyte stimulation and release of many inflammatory mediators like cytokines, growth factors, components of the complement system, acute phase proteins, and other immunoregulators. Due to the potential applications of this therapy, many studies have been reported regarding the effect of the treatment on cell survival/death, cell proliferation, matrix assembly, proteases and inhibitors, among others. Studies have demonstrated that PDT alters the extracellular matrix profoundly. For example, PDT induces collagen matrix changes, including cross-linking. The extracellular matrix is vital for tissue organization in multicellular organisms. In cooperation with growth factors and cytokines, it provides cells with key signals in a variety of physiological and pathological processes, for example, adhesion/migration and cell proliferation/differentiation/death. Thus, the focus of the present paper is related to the effects of PDT observed on the extracellular matrix and on the molecules associated with it, such as, adhesion molecules, matrix metalloproteinases, growth factors, and immunological mediators.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53%), TIMP-1 (-31.7%), TGF-β1 (-57.7%), and MMP-2 (-41.6%), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1%), ALT (-17.6%), and AST (-12.2%) serum levels; c) increased liver weight (11.3%), and reduced liver collagen (-37.1%), regenerative nodules size (-22.1%), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and “re-couples” NOS function.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Role of the extracellular matrix in variations of invasive pathways in lung cancers

Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas (SqCC), is the massive degradation of the extracellular matrix. The remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan synthases (HAS), E-cadherin adhesion molecules, and the transforming growth factor β (TGF-β) may favor invasion, cellular motility, and proliferation. We examined HAase proteins (Hyal), HAS, E-cadherin, and TGF-β profiles in lung AD subtypes and SqCC obtained from smokers and non-smokers. Fifty-six patients, median age 64 years, who underwent lobectomy for AD (N = 31) and SqCC (N = 25) were included in the study. HAS-1, -2 and -3, and Hyal-1 and -3 were significantly more expressed by tumor cells than normal and stroma cells (P < 0.01). When stratified according to histologic types, HAS-3 and Hyal-1 immunoreactivity was significantly increased in tumor cells of AD (P = 0.01) and stroma of SqCC (P = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS-3 immunoreactivity in tumor cells (P < 0.01). Stroma cells of SqCC from non-smokers presented a significant association with HAS-3 (P < 0.01). Hyal, HAS, E-cadherin, and TGF-β modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. HAases in resected AD and SqCC were strongly related to the prognosis. Therefore, our findings suggest that strategies aimed at preventing high HAS-3 and Hyal-1 synthesis, or local responses to low TGF-β and E-cadherin, may have a greater impact in lung cancer prognosis.

Extracellular vesicles: structure, function, and potential clinical uses in renal diseases

Interest in the role of extracellular vesicles in various diseases including cancer has been increasing. Extracellular vesicles include microvesicles, exosomes, apoptotic bodies, and argosomes, and are classified by size, content, synthesis, and function. Currently, the best characterized are exosomes and microvesicles. Exosomes are small vesicles (40-100 nm) involved in intercellular communication regardless of the distance between them. They are found in various biological fluids such as plasma, serum, and breast milk, and are formed from multivesicular bodies through the inward budding of the endosome membrane. Microvesicles are 100-1000 nm vesicles released from the cell by the outward budding of the plasma membrane. The therapeutic potential of extracellular vesicles is very broad, with applications including a route of drug delivery and as biomarkers for diagnosis. Extracellular vesicles extracted from stem cells may be used for treatment of many diseases including kidney diseases. This review highlights mechanisms of synthesis and function, and the potential uses of well-characterized extracellular vesicles, mainly exosomes, with a special focus on renal functions and diseases.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

Prodrug strategies of antiviral nucleotides: Studies on enzymatically and thermally removable phosphate protecting groups

Antiviral nucleosides are compounds that are used against viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). To act as therapeutic agent, the antiviral nucleoside needs to be phosphorylated to nucleotide in the body in three consecutive phosphorylation steps by cellular or viral enzymes. The first phosphorylation to the nucleoside monophosphate is often inefficient and leads to poor antiviral activity. The antiviral efficacy can be improved by applying a prodrug strategy and delivering the antiviral nucleoside directly as its monophosphate. In prodrug strategies of antiviral nucleotides, the negative charges on the phosphate moiety are temporarily masked with protecting groups. Once inside the cell, the protecting groups are removed by enzymatic or chemical processes. Many prodrug strategies apply biodegradable protecting groups, the removal of which is triggered by esterase enzymes. Several studies have, however, demonstrated that the removal rate of the second and subsequent esterase labile protecting groups significantly slows down after the first protecting group is removed due to the negative charge on the phosphodiester intermediate, which disturbs the catalytic site of the enzyme. In this thesis, esterase labile protecting group strategies where the issue of retardation could be avoided were studied. Prodrug candidates of antiviral nucleotides were synthesized and kinetic studies on the chemical and enzymatic stability were carried out. In the synthesized compounds, the second protecting group is cleaved from the monophosphate some other mechanism than esterase triggered activation or the structure of prodrug requires only one protecting group. In addition, esterase labile protecting group which is additionally thermally removable was studied. This protecting group was cleaved from oligomeric phosphodiesters both enzymatically and thermally and seems most attractive of the studied phosphate protecting groups. However, the rate of the thermal removal still is too slow to allow efficient protection of longer oligonucleotides and needs optimization. Key words: antiviral, nucleotide, prodrug, protecting group, biodegradable

The role of extracellular matrix macromolecules in cancer and diabetic macroangiopathy – with special reference to decorin and hyaluronan

The central role of extracellular matrix (ECM) macromolecules in diseases such as cancer and atherosclerotic vascular diseases including diabetic macroangiopathy is indisputable. Decorin and hyaluronan (HA) represent vital ECM macromolecules in the microenvironment of cells and are centrally involved in human cancer and cardiovascular biology. In cancer, decorin is considered to play a tumor suppressive role. However, there is some discrepancy whether malignant cells express it. Regarding HA, its contribution to the development of atherosclerotic vascular diseases has been well established. Nevertheless, the precise role of HA in arterial narrowing associated with diabetes is not known. The present study focused on two vital ECM macromolecules, namely decorin and HA. First, decorin expression was studied in human tumorigenesis. Furthermore, the effect of adenovirus-mediated decorin transduction on selected cancer cell lines was investigated. The results invariably showed that cancer cells completely lacked decorin expression. The study also demonstrated that transducing cancer cells with decorin adenoviral vector markedly inhibited their malignant behavior. In line with this, a strong induction of decorin expression in normal human embryonic stem cells (hESCs), but not in abnormal hESCs was observed during their differentiation. Secondly, the significance of HA in the development of diabetic macroangiopathy in response to hyperglycemia was evaluated. Results showed that the synthesis of HA by vascular smooth muscle cells was significantly increased in response to high glucose concentration. This increase was associated with the diminished ability of the cells to contract collagen-rich matrix suggesting that HA participates in the disturbed vascular remodeling of diabetic patients. The results of this study support endeavours to develop novel ECM macromolecule -based therapies targeting cancer and cardiovascular diseases.