Viral Entry Inhibitors Protect against SARS-CoV-2-Induced Neurite Shortening in Differentiated SH-SY5Y Cells.

The utility of human neuroblastoma cell lines as in vitro model to study neuro-invasiveness and neuro-virulence of SARS-CoV-2 has been demonstrated by our laboratory and others. The aim of this report is to further characterize the associated cellular responses caused by a pre-alpha SARS-CoV-2 strain on differentiated SH-SY5Y and to prevent its cytopathic effect by using a set of entry inhibitors. The susceptibility of SH-SY5Y to SARS-CoV-2 was confirmed at high multiplicity-of-infection, without viral replication or release. Infection caused a reduction in the length of neuritic processes, occurrence of plasma membrane blebs, cell clustering, and changes in lipid droplets electron density. No changes in the expression of cytoskeletal proteins, such as tubulins or tau, could explain neurite shortening. To counteract the toxic effect on neurites, entry inhibitors targeting TMPRSS2, ACE2, NRP1 receptors, and Spike RBD were co-incubated with the viral inoculum. The neurite shortening could be prevented by the highest concentration of camostat mesylate, anti-RBD antibody, and NRP1 inhibitor, but not by soluble ACE2. According to the degree of entry inhibition, the average amount of intracellular viral RNA was negatively correlated to neurite length. This study demonstrated that targeting specific SARS-CoV-2 host receptors could reverse its neurocytopathic effect on SH-SY5Y.

Volumetry of ovine incisors dental pulp for further regenerative therapy.

Mesenchymal stem cells (MSCs) are used for regenerative therapy. Dental pulp MSCs make extracted wisdom teeth a useful resource in humans. Preclinical validation of regenerative therapies requires large animal models such as the sheep. Since stem cells can be retrieved from the dental pulp of ovine incisors, the best age to extract a maximal volume of dental pulp needs to be defined. The objective of this ex vivo study was to quantify incisors dental pulp volume, in sheep of various age. Three jaws were dedicated to histology (one per age group); the others were imaged with a computed tomography scanner [3 years-old (n = 9), 4 (n = 3) and 6 (n = 5)]. The incisors dental pulp volume was measured after 3D reconstruction. Multiple linear regression showed that dental pulp volume of ovine incisors decreases with age (ß-estimate = -3.3; p < 0.0001) and teeth position from the more central to the more lateral (ß-estimate = -4.9; p = 0.0009). Weight was not a relevant variable in the regression model. The dental pulp volume ranged from 36.7 to 19.6 mm3 in 3-year-old sheep, from 23.6 to 11.3 in 4-year-old sheep, and from 19.4 to 11.5 in 6-year-old sheep. The pulp volume of the most central teeth (first intermediate) was significantly higher than the most lateral teeth (corner). Haematoxylin-Eosin-Safran of the whole incisors, and of isolated dental pulps demonstrated a similar morphology to that in humans. The first intermediate incisor of 3-year-old sheep should be selected preferentially in preclinical research to retrieve the highest volume of dental pulp.

Chronic Sulfasalazine Treatment in Mice Induces System xc - - Independent Adverse Effects.

Despite ample evidence for the therapeutic potential of inhibition of the cystine/glutamate antiporter system xc - in neurological disorders and in cancer, none of the proposed inhibitors is selective. In this context, a lot of research has been performed using the EMA- and FDA-approved drug sulfasalazine (SAS). Even though this molecule is already on the market for decades as an anti-inflammatory drug, serious side effects due to its use have been reported. Whereas for the treatment of the main indications, SAS needs to be cleaved in the intestine into the anti-inflammatory compound mesalazine, it needs to reach the systemic circulation in its intact form to allow inhibition of system xc -. The higher plasma levels of intact SAS (or its metabolites) might induce adverse effects, independent of its action on system xc -. Some of these effects have however been attributed to system xc - inhibition, calling into question the safety of targeting system xc -. In this study we chronically treated system xc - - deficient mice and their wildtype littermates with two different doses of SAS (160 mg/kg twice daily or 320 mg/kg once daily, i.p.) and studied some of the adverse effects that were previously reported. SAS had a negative impact on the survival rate, the body weight, the thermoregulation and/or stress reaction of mice of both genotypes, and thus independent of its inhibitory action on system xc -. While SAS decreased the total distance travelled in the open-field test the first time the mice encountered the test, it did not influence this parameter on the long-term and it did not induce other behavioral changes such as anxiety- or depressive-like behavior. Finally, no major histological abnormalities were observed in the spinal cord. To conclude, we were unable to identify any undesirable system xc --dependent effect of chronic administration of SAS.

Cystine-glutamate antiporter deletion accelerates motor recovery and improves histological outcomes following spinal cord injury in mice.

xCT is the specific subunit of System xc-, an antiporter importing cystine while releasing glutamate. Although xCT expression has been found in the spinal cord, its expression and role after spinal cord injury (SCI) remain unknown. The aim of this study was to characterize the role of xCT on functional and histological outcomes following SCI induced in wild-type (xCT+/+) and in xCT-deficient mice (xCT-/-). In the normal mouse spinal cord, slc7a11/xCT mRNA was detected in meningeal fibroblasts, vascular mural cells, astrocytes, motor neurons and to a lesser extent in microglia. slc7a11/xCT gene and protein were upregulated within two weeks post-SCI. xCT-/- mice recovered muscular grip strength as well as pre-SCI weight faster than xCT+/+ mice. Histology of xCT-/- spinal cords revealed significantly more spared motor neurons and a higher number of quiescent microglia. In xCT-/- mice, inflammatory polarization shifted towards higher mRNA expression of ym1 and igf1 (anti-inflammatory) while lower levels of nox2 and tnf-a (pro-inflammatory). Although astrocyte polarization did not differ, we quantified an increased expression of lcn2 mRNA. Our results show that slc7a11/xCT is overexpressed early following SCI and is detrimental to motor neuron survival. xCT deletion modulates intraspinal glial activation by shifting towards an anti-inflammatory profile.

Susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2 infection.

Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes - needed for invading epithelial cells - were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl. Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

Shedding of Brucella melitensis happens through milk macrophages in the murine model of infection.

Although shedding of zoonotic brucellae in milk has been demonstrated in natural hosts, these data are still missing for the standard murine infection model. We therefore analysed shedding kinetics and the niche of B. melitensis in murine milk. Pregnant Balb/cByJ mice were intraperitoneally infected with 105 CFU of the 16 M reference strain, a 16 M mCherry mutant or a human isolate. Milk was collected over the course of lactation, and subjected to culture and immunofluorescence assays. Bacteria were also quantified in spleen and mammary glands of maternal mice and in spleen of the litter. The shedding of the three strains did not differ significantly (p = 0.301), ranging from log10 1.5 to 4.04 CFU/ml. A total of 73% of the mice excreted B. melitensis into the milk with peak values at mid-lactation; up to 30 bacteria/cell were found in macrophages and neutrophils. While the bacterial counts in the spleen of lactating females confirmed a well-established infection, only 50% of the pups harboured brucellae in their spleen, including the spleen of an uninfected pup fed by an infected foster mother. In conclusion, the murine model of infection may contribute to a better understanding of the zoonotic transmission of brucellosis.

Phagocyte-specific S100A8/A9 is upregulated in primary Sjögren's syndrome and triggers the secretion of pro-inflammatory cytokines in vitro.

OBJECTIVES: To determine the role of S100A8/A9 in the pathogenesis of primary Sjögren's syndrome (pSS). METHODS: The serum levels of S100A8/A9 were determined in pSS patients and healthy controls by ELISA. The expression of S100A8/A9 in salivary glands was assessed by immunohistochemistry. The phenotype of S100A8+ and S100A9+ cells was identified using double immunofluorescence. The effects of S100A8/A9 on cytokine production by peripheral blood mononuclear cells (PBMCs) from pSS patients were determined in vitro by flow cytometry. The effects of pro-inflammatory cytokines on S100A8/A9 secretion were additionally investigated in vitro by ELISA in PBMCs from pSS patients and control subjects. RESULTS: Serum levels of S100A8/A9 were significantly increased in pSS patients compared to healthy controls. The tissular expression of S100A8 and S100A9, identified in professional phagocytes (neutrophils, monocytes and plasmacytoid dendritic cells), was increased in the salivary glands of pSS patients and correlated with focus score. In vitro, recombinant S100A8/A9 increased the production of IL-1ß, IL-6, TNF-α, IFN-γ, IL-10, IL-17A and IL-22 by PBMCs. The S100A8/A9-induced increase in TNF-α production in pSS patients was significant relative to controls. Furthermore, IL-1ß, TNF-α, IL-6, and IL-17A stimulated release of S100A8/A9 from PBMCs in pSS patients. CONCLUSIONS: S100A8/A9 is increased in pSS patients contributing to the in vitro increased production of pro-inflammatory cytokines. As such, S100A8/A9 in concert with other cytokines might contribute to the pathogenesis of pSS.

Modelling and treating amyotrophic lateral sclerosis through induced-pluripotent stem cells technology.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease affecting primarily the population of motor neurons, even though a non-cell autonomous component, involving neighbouring non-neuronal cells, is more and more described. Despite 140 years of disease experience, still no efficient treatment exists against ALS. The inability to readily obtain the faulty cell types relevant to ALS has impeded progress in drug discovery for decades. However, the pioneer work of Shinya Yamanaka in 2007 in the stem cell field was a real breakthrough. Recent advances in cell reprogramming now grant access to significant quantities of CNS disease-affected cells. Induced pluripotent stem cells (iPSc) have been recently derived from patients carrying mutations linked to familial forms of ALS as well as from sporadic patients. Precise and mature protocols allow now their differentiation into ALS-relevant cell subtypes; sustainable and renewable sources of human motor neurons or glia are being available for ALS disease modelling, drug screening or for the development of cell therapies. In few years, the proof-of-concept was made that ALS disease-related phenotypes can be reproduced with iPSc and despite some remaining challenges, we are now not so far to provide platforms for the investigation of ALS therapeutics. This paper also reviews the pioneering studies regarding the applicability of iPSc technology in ALS animal models. From modest slowing down of ALS progression to no severe adverse effects, iPSc-based cell therapy resulted in promising premises in ALS preclinical paradigms, although long-term surveys are highly recommended.

Transplantation of stem cell-derived astrocytes for the treatment of amyotrophic lateral sclerosis and spinal cord injury.

Neglected for years, astrocytes are now recognized to fulfill and support many, if not all, homeostatic functions of the healthy central nervous system (CNS). During neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI), astrocytes in the vicinity of degenerating areas undergo both morphological and functional changes that might compromise their intrinsic properties. Evidence from human and animal studies show that deficient astrocyte functions or loss-of-astrocytes largely contribute to increased susceptibility to cell death for neurons, oligodendrocytes and axons during ALS and SCI disease progression. Despite exciting advances in experimental CNS repair, most of current approaches that are translated into clinical trials focus on the replacement or support of spinal neurons through stem cell transplantation, while none focus on the specific replacement of astroglial populations. Knowing the important functions carried out by astrocytes in the CNS, astrocyte replacement-based therapies might be a promising approach to alleviate overall astrocyte dysfunction, deliver neurotrophic support to degenerating spinal tissue and stimulate endogenous CNS repair abilities. Enclosed in this review, we gathered experimental evidence that argue in favor of astrocyte transplantation during ALS and SCI. Based on their intrinsic properties and according to the cell type transplanted, astrocyte precursors or stem cell-derived astrocytes promote axonal growth, support mechanisms and cells involved in myelination, are able to modulate the host immune response, deliver neurotrophic factors and provide protective molecules against oxidative or excitotoxic insults, amongst many possible benefits. Embryonic or adult stem cells can even be genetically engineered in order to deliver missing gene products and therefore maximize the chance of neuroprotection and functional recovery. However, before broad clinical translation, further preclinical data on safety, reliability and therapeutic efficiency should be collected. Although several technical challenges need to be overcome, we discuss the major hurdles that have already been met or solved by targeting the astrocyte population in experimental ALS and SCI models and we discuss avenues for future directions based on latest molecular findings regarding astrocyte biology.

Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.

AIM: Biomarker-based tracking of human stem cells xenotransplanted into animal models is crucial for studying their fate in the field of cell therapy or tumor xenografting. MATERIALS & METHODS: Using immunohistochemistry and in situ hybridization, we analyzed the expression of three human-specific biomarkers: Ku80, human mitochondria (hMito) and Alu. RESULTS: We showed that Ku80, hMito and Alu biomarkers are broadly expressed in human tissues with no or low cross-reactivity toward rat, mouse or pig tissues. In vitro, we demonstrated that their expression is stable over time and does not change along the differentiation of human-derived induced pluripotent stem cells or human glial-restricted precursors. We tracked in vivo these cell populations after transplantation in rodent spinal cords using aforementioned biomarkers and human-specific antibodies detecting apoptotic, proliferating or neural-committed cells. CONCLUSION: This study assesses the human-species specificity of Ku80, hMito and Alu, and proposes useful biomarkers for characterizing human stem cells in xenotransplantation paradigms.

Overexpression of the astrocyte glutamate transporter GLT1 exacerbates phrenic motor neuron degeneration, diaphragm compromise, and forelimb motor dysfunction following cervical contusion spinal cord injury.

A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP(+) astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.

Neural progenitors derived from human induced pluripotent stem cells survive and differentiate upon transplantation into a rat model of amyotrophic lateral sclerosis.

Human induced pluripotent stem cells (iPSCs) offer hope for personalized regenerative cell therapy in amyotrophic lateral sclerosis (ALS). We analyzed the fate of human iPSC-derived neural progenitors transplanted into the spinal cord of wild-type and transgenic rats carrying a human mutated SOD1(G93A) gene. The aim was to follow survival and differentiation of human neural progenitors until day 60 post-transplantation in two different in vivo environments, one being ALS-like. iPSC-derived neural progenitors efficiently engrafted in the adult spinal cord and survived at high numbers. Different neural progenitor, astroglial, and neuronal markers indicated that, over time, the transplanted nestin-positive cells differentiated into cells displaying a neuronal phenotype in both wild-type and transgenic SOD1 rats. Although a transient microglial phenotype was detected at day 15, astroglial staining was negative in engrafted cells from day 1 to day 60. At day 30, differentiation toward a neuronal phenotype was identified, which was further established at day 60 by the expression of the neuronal marker MAP2. A specification process into motoneuron-like structures was evidenced in the ventral horns in both wild-type and SOD1 rats. Our results demonstrate proof-of-principle of survival and differentiation of human iPSC-derived neural progenitors in in vivo ALS environment, offering perspectives for the use of iPSC-based therapy in ALS.

Glucose transport by acinar cells in rat parotid glands.

BACKGROUND/AIMS: Salivary glucose is often considered as being from glandular origin. Little information is available, however, on the possible role of glucose transporters in the secretion of the hexose by salivary glands. The major aim of the present study was to investigate the expression and localization of several distinct glucose transporters in acinar cells of rat parotid glands. METHODS: Quantitative real-time PCR analysis, immunohistochemistry and western blotting techniques were used to assess the presence of SGLT1, GLUT1, GLUT2 and GLUT4 in acinar cells of rat parotid glands. RESULTS: Quantitative real-time PCR documented the expression of SGLT1 and GLUT1 in parotid tissues, with a much lower level of GLUT4 mRNA and no expression of GLUT2 mRNA. Western blot analysis revealed the presence of SGLT1, GLUT1 and GLUT4 proteins, but not GLUT2 proteins in the parotid extract. Immunohistochemistry confirmed these findings. SGLT1 was specifically located at the baso-lateral membrane, co-localizing with Na(+)/K(+) ATPase. GLUT1 was found both at the baso-lateral and apical level. GLUT4 appeared to be also located at the baso-lateral level. However, too little GLUT4 was present to allow co-localization labeling. CONCLUSION: Based on these findings, a model is proposed for the transport of glucose into the acinar cells and thereafter into the acinar lumen.

Levels of kinesin light chain and dynein intermediate chain are reduced in the frontal cortex in Alzheimer's disease: implications for axoplasmic transport.

Fast anterograde and retrograde axoplasmic transports in neurons rely on the activity of molecular motors and are critical for maintenance of neuronal and synaptic functions. Disturbances of axoplasmic transport have been identified in Alzheimer's disease and in animal models of this disease, but their mechanisms are not well understood. In this study we have investigated the distribution and the level of expression of kinesin light chains (KLCs) (responsible for binding of cargos during anterograde transport) and of dynein intermediate chain (DIC) (a component of the dynein complex during retrograde transport) in frontal cortex and cerebellar cortex of control subjects and Alzheimer's disease patients. By immunoblotting, we found a significant decrease in the levels of expression of KLC1 and 2 and DIC in the frontal cortex, but not in the cerebellar cortex, of Alzheimer's disease patients. A significant decrease in the levels of synaptophysin and of tubulin-ß3 proteins, two neuronal markers, was also observed. KLC1 and DIC immunoreactivities did not co-localize with neurofibrillary tangles. The mean mRNA levels of KLC1, 2 and DIC were not significantly different between controls and AD patients. In SH-SY5Y neural cells, GSK-3ß phosphorylated KLC1, a change associated to decreased association of KLC1 with its cargoes. Increased levels of active GSK-3ß and of phosphorylated KLC1 were also observed in AD frontal cortex. We suggest that reduction of KLCs and DIC proteins in AD cortex results from both reduced expression and neuronal loss, and that these reductions and GSK-3ß-mediated phosphorylation of KLC1 contribute to disturbances of axoplasmic flows and synaptic integrity in Alzheimer's disease.

Genetically modified stem cells for the treatment of neurological diseases.

The central nervous system has a very poor regenerative potential and is difficult to access. This partly explains why neurological diseases often lack appropriate therapeutic options and represent the most significant burden for healthcare systems. Progress in understanding the molecular background of neurological diseases requires innovative approaches offering new hope for the patients. One of the most intriguing and promising options is the combination of stem cells with gene therapy. Unlike fibroblasts, stem cells exhibit a high tropism for disease-affected tissue and integrate into the nervous tissue. This makes them ideal candidates for the production and delivery of molecules of interest for treating the nervous system. This article reviews the methodology for obtaining pluripotent stem cells (iPSCs) as precursors for neuronal cells, glial cells and the current state of the art in applications of genetically modified stem cells in animal models of neurodegenerative diseases, stroke, axonal damage, tumors and epilepsy.

Distribution, differentiation, and survival of intravenously administered neural stem cells in a rat model of amyotrophic lateral sclerosis.

The transplantation of neural stem cells (NSCs) is a challenging therapeutic strategy for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). To provide insight into the potential of the intravenous delivery of NSCs, we evaluated the delivery of NSCs marked with green fluorescent protein to the central nervous system (CNS) via intravenous tail vein injections in an ALS model. The injected cell fates were followed 1, 3, and 7 days after transplantation. The highest efficiency of cell delivery to the CNS was found in symptomatic ALS (up to 13%), moderate in presymptomatic ALS (up to 6%), and the lowest in wild-type animals (up to 0.3%). NSCs injected into ALS animals preferentially colonized the motor cortex, hippocampus, and spinal cord, and their differentiation was characterized by a decrease of nestin expression and the appearance of MAP2-, GFAP-, O4-, and CD68-positive cells. Tumor necrosis factor (TNF) administration increased the CNS delivery of transplanted cells in wild-type and presymptomatic, but not ALS symptomatic animals. Moreover, a TNF-related increase in NSC differentiation and survival was detected. Apoptosis was detected as the main cause of the loss of transplanted cells and it was influenced by TNF. Although 3 days after TNF treatment cell death was accelerated, TNF slowed down apoptosis after 7 days. This study provides elementary facts about the process occurring after NSCs leave the blood stream and enter the nervous tissue affected by inflammation/degeneration, which should help facilitate the planning of future bench-to-bedside translational projects.

Gemals, a new drug candidate, extends lifespan and improves electromyographic parameters in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease involving selective and progressive degeneration and death of motor neurons. ALS is a multifactorial disease in which oxidative stress, glutamate excitotoxicity, intracellular aggregates, neurofilamentous disorganization, zinc excitotoxicity, mitochondrial damage, neuroinflammation, abnormalities in growth factors and apoptosis play a role. Any therapeutic approach to delay or stop the evolution of ALS should therefore ideally target these multiple pathways leading to motor neuron death. We have developed a combination therapy (Gemals) composed of functional polypeptides (fatty acids, free radical scavengers and amino acids linked to poly-L-lysine), chosen according to their known potentiality for regeneration or protection of neuronal components such as myelin, axon transport and mitochondria. We found that Gemals significantly extended lifespan and improved electromyographic parameters in a SOD1(G93A) rat model. The use of two drug concentrations indicated a possible dose dependence. These initial findings open the way to further investigation necessary to validate this new drug as a candidate for ALS treatment.

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa.

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.