Continuous cultivation of human hamstring tenocytes on microcarriers in a spinner flask bioreactor system

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering‐based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon‐derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 × 10⁶ cells/1000 cm² surface) on Cytodex? type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD‐PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead‐to‐bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:142–151, 2014     

Activated protein C: A regulator of human skin epidermal keratinocyte function

Activated protein C(APC) is a physiological anticoagulant, derived from its precursor protein C(PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor(EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.     

Generation of an antibody with a designed specificity difference for protein C and activated protein C

Rabbit polyclonal antibodies to a synthetic peptide, NH₂-Asp-Thr-Asn-Gln-Val-Asp-Gln-Lys-Asp-Gln-Leu-Asp-Phe-Arg-CONH₂ (APep), have been produced. This sequence is identical to that contained in the tetradecapeptide released from bovine protein C (PC) as a result of its conversion to its activated form (APC), except that Phe₁₃ replaced the normal Pro₁₃, in order to discourage cross-reactivity of antibodies to the carboxylterminal portion of APep with PC. The antibody pool obtained reacted with PC and showed virtually no cross-reactivity toward either APC or several typical plasma proteins. This general approach should serve well as a means of production of antibodies with a designed specificity capable of distinguishing between forms of the same protein that arise by release of peptide material.     

Activated protein C via PAR1 receptor regulates survival of neurons under conditions of glutamate excitotoxicity

The effect of an anticoagulant and cytoprotector blood serine proteinase--activated protein C (APC)--on survival of cultured hippocampal and cortical neurons under conditions of glutamate-induced excitotoxicity has been studied. Low concentrations of APC (0.01-10 nM) did not cause neuron death, but in the narrow range of low concentrations APC twofold and stronger decreased cell death caused by glutamate toxicity. High concentrations of APC (>50 nM) induced the death of hippocampal neurons similarly to the toxic action of glutamate. The neuroprotective effect of APC on the neurons was mediated by type 1 proteinase-activated receptor (PAR1), because the inactivation of the enzyme with phenylmethylsulfonyl fluoride or PAR1 blockade by a PAR1 peptide antagonist ((Tyr1)-TRAP-7) prevented the protective effect of APC. Moreover, APC inhibited the proapoptotic effect of 10 nM thrombin on the neurons. Geldanamycin, a specific inhibitor of heat shock protein Hsp90, completely abolished the antiapoptotic effect of 0.1 nM APC on glutamate-induced cytotoxicity in the hippocampal neurons. Thus, APC at low concentrations, activating PAR1, prevents the death of hippocampal and cortical neurons under conditions of glutamate excitotoxicity.     

Activated protein C--an anticoagulant that does more than stop clots

Activated protein C (APC) is a glycoprotein derived from its precursor, protein C and formed by the cleavage of an activation peptide by thrombin bound to thrombomodulin. Originally thought to be synthesized exclusively by the liver, recent reports have shown that protein C is synthesized by endothelial cells, keratinocytes and some hematopoietic cells.APC functions as a physiological anticoagulant with cytoprotective, anti-inflammatory and anti-apoptotic properties. In vitro and preclinical data have revealed that APC exerts its protective effects via an intriguing mechanism requiring endothelial protein C receptor and the thrombin receptor, protease-activated receptor-1. Remarkably, even though APC cleaves this receptor in an identical fashion to thrombin, it exerts opposing effects.Recently approved as a therapeutic agent for severe sepsis, APC is now emerging as a potential treatment for a number of autoimmune and inflammatory diseases including lung disorders, spinal cord injury and chronic wounds. The future pharmacologic use of APC holds remarkable promise.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

Cooling therapy upregulates protein C activation in heat stressed rats: An experimental study

Protein C (PC) pathway homeostasis is implicated in heat stress (HS). This study determines whether cooling could improve the PC pathway in HS. Fifty-six anesthetized rats were warmed to achieve HS (rectal temperature [Tr] 42°C). These rats were divided into seven groups: (a) control group:sacrifice immediately 15 min after HS; (b) HS+I:sacrifice immediately after 15 min ice-water treatment or (c) 3 hr after HS; (d) HS+C:sacrifice immediately after 15-min cold-water treatment or (e) 3 hr after HS; (f) HS: sacrifice immediately 15 min after HS or (g) 3 hr after HS. Plasma PC, activated protein C (APC), and soluble thrombomodulin (sTM) levels were tested at both time points. After cooling, Tr in the HS+I and HS+C groups significantly decreased, when compared with the HS group, and Tr was significantly lower in the HS+I group than in the HS+C group ( p < 0.05). Furthermore, sTM levels were highest in the HS group among the groups at both time points. Plasma PC and APC levels increased after HS. In the HS+I and HS+C groups, plasma APC levels and the APC/PC ratio significantly increased at both time points. The proportions were significantly higher in the HS+I group than in the HS+C group, and there was no significant increase in APC/PC ratio in the HS group. Cooling exerts an anticoagulant effect following HS by increasing APC levels. Ice-water blanket therapy is more effective than cold-water blanket therapy in increasing APC levels.     

Down-regulation of endothelial protein C receptor promotes preeclampsia by affecting actin polymerization

Preeclampsia is a severe pregnancy-related disease that is found in 3%–5% of pregnancies worldwide and is primarily related to the decreased proliferation and invasion of trophoblast cells and abnormal uterine spiral artery remodelling. However, studies on the pathogenesis of placental trophoblasts are insufficient, and the aetiology of PE remains unclear. Here, we report that endothelial protein C receptor (EPCR), a transmembrane glycoprotein, was down-regulated in placentas from preeclamptic patients. Moreover, lack of EPCR significantly reduced the trophoblast cell proliferation, invasion and tube formation capabilities. Microscale thermophoresis analysis showed that EPCR directly bound to protease-activated receptor 1 (PAR-1), a G protein-coupled receptor. This change resulted in a substantial reduction in active Rac1 and caused excessive actin rearrangement. Our findings reveal a previously unidentified role of EPCR in the regulation of trophoblast proliferation, invasion and tube formation through promotion of actin polymerization, which is required for normal placental development.     

RACK1, a PKC targeting protein, is exclusively localized to basal airway epithelial cells.

The novel isoform of protein kinase C (PKC), PKCepsilon, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCepsilon signaling in the airways and the potential role of the PKCepsilon-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, beta-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCepsilon signaling in the basal airway cell may involve RACK1; however, PKCepsilon regulation in ciliated cells uses RACK1-independent pathways.     

The anticoagulant protein C pathway

The anticoagulant protein C system regulates the activity of coagulation factors VIIIa and Va, cofactors in the activation of factor X and prothrombin, respectively. Protein C is activated on endothelium by the thrombin-thrombomodulin-EPCR (endothelial protein C receptor) complex. Activated protein C (APC)-mediated cleavages of factors VIIIa and Va occur on negatively charged phospholipid membranes and involve protein cofactors, protein S and factor V. APC also has anti-inflammatory and anti-apoptotic activities that involve binding of APC to EPCR and cleavage of PAR-1 (protease-activated receptor-1). Genetic defects affecting the protein C system are the most common risk factors of venous thrombosis. The protein C system contains multi-domain proteins, the molecular recognition of which will be reviewed.     

Endothelial protein C receptor is expressed in rat cortical and hippocampal neurons and is necessary for protective effect of activated protein C at glutamate excitotoxicity

Activated protein C (APC) is an anticoagulant and anti-inflammatory factor that acts via endothelial protein C receptor (EPCR). Interestingly, APC also exhibits neuroprotective activities. In the present study, we demonstrate for the first time expression of EPCR, the receptor for APC, in rat cortical and hippocampal neurons. Moreover, exposing the neurons to glutamate excitotoxicity we studied the functional consequence of the expression of EPCR. By cytotoxicity assay we showed that EPCR was necessary for the APC-mediated protective effect in both neuronal cell types in culture. The effect of APC was abrogated in the presence of blocking EPCR antibodies. Analysis of neuronal death by cell labelling with dyes which allow distinguishing living and dead cells confirmed that the anti-apoptotic effect of APC was dependent on both EPCR and protease-activated receptor-1. Thus, we suggest that binding of APC to EPCR on neurons and subsequent activation of protease-activated receptor-1 by the complex of APC-EPCR promotes survival mechanisms after exposure of neurons to damaging factors.     

High endogenous activated protein C levels attenuates bleomycin‐induced pulmonary fibrosis

Coagulation activation accompanied by reduced anticoagulant activity is a key characteristic of patients with idiopathic pulmonary fibrosis (IPF). Although the importance of coagulation activation in IPF is well studied, the potential relevance of endogenous anticoagulant activity in IPF progression remains elusive. We assess the importance of the endogenous anticoagulant protein C pathway on disease progression during bleomycin‐induced pulmonary fibrosis. Wild‐type mice and mice with high endogenous activated protein C APC levels (APC^high) were subjected to bleomycin‐induced pulmonary fibrosis. Fibrosis was assesses by hydroxyproline and histochemical analysis. Macrophage recruitment was assessed immunohistochemically. In vitro, macrophage migration was analysed by transwell migration assays. Fourteen days after bleomycin instillation, APC^high mice developed pulmonary fibrosis to a similar degree as wild‐type mice. Interestingly, Aschcroft scores as well as lung hydroxyproline levels were significantly lower in APC^high mice than in wild‐type mice on day 28. The reduction in fibrosis in APC^high mice was accompanied by reduced macrophage numbers in their lungs and subsequent in vitro experiments showed that APC inhibits thrombin‐dependent macrophage migration. Our data suggest that high endogenous APC levels inhibit the progression of bleomycin‐induced pulmonary fibrosis and that APC modifies pulmonary fibrosis by limiting thrombin‐dependent macrophage recruitment.     

Effects of protein kinase C modulation on NMDA receptor mediated regulation of neurotransmitter enzyme and c-fos protein in cultured neurons

The role of protein kinase C (PKC) in N-methyl-d-aspartate (NMDA) receptor-mediated biochemical differentiation and c-fos protein expression was investigated in cultured cerebellar granule neurons. The biochemical differentiation of glutamatergic granule cells was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 1 to 3 days (between 2 and 5 days in vitro), it elevated the specific activity of glutaminase. In contrast, NMDA had little effect on the activity of aspartate aminotransferase or of lactate dehydrogenase. Treatment of 10-day old granule neurons with NMDA also resulted in a marked increase in the immunocytochemically measured expression of c-fos protein. The increases in both the activity of glutaminase and the steady state level of c-fos protein were specific to the activation of NMDA receptors, as they were completely blocked byd,l-2-amino-5-phosphonovaleric acid. The specific stimulation of NMDA receptors in PKC-depleted granule neurons or in the presence of reasonably specific PKC inhibitors also produced significant elevation in the activity of glutaminase and the expression of c-fos protein. These increases were similar in magnitude to those observed in the granule neurons of the respective control groups. Our findings demonstrate that PKC is not directly involved in the NMDA receptor-mediated signal transduction processes associated with biochemical differentiation and c-fos induction in cerebellar granule neurons.     

AMP-activated protein kinase mediates VEGF-stimulated endothelial NO production

Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell function. VEGF stimulates NO production, proposed to be a result of phosphorylation and activation of endothelial NO synthase (eNOS) at Ser1177. Phosphorylation of eNOS at this site also occurs after activation of AMP-activated protein kinase (AMPK) in cultured endothelial cells. We therefore determined whether AMPK mediates VEGF-stimulated NO synthesis in endothelial cells. VEGF caused a rapid, dose-dependent stimulation of AMPK activity, with a concomitant increase in phosphorylation of eNOS at Ser1177. Infection of endothelial cells with an adenovirus expressing a dominant negative mutant AMPK partially inhibited both VEGF-stimulated eNOS Ser1177 phosphorylation and NO production. VEGF-stimulated AMPK activity was completely inhibited by the Ca(2+)/calmodulin-dependent protein kinase kinase inhibitor, STO-609. Stimulation of AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase represents a novel signalling mechanism utilised by VEGF in endothelial cells that contributes to eNOS phosphorylation and NO production.     

Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M_r), KP-2 (pI 4.7/20,700 M_r) and KP-10 (pI 4.7/25,000 M_r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca²⁺-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca²⁺-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.     

Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.