Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

Fibroblast-derived Extracellular Vesicles Induce Lung Cancer Progression in the Idiopathic Pulmonary Fibrosis Microenvironment.

Idiopathic pulmonary fibrosis (IPF) is a progressive aging-related lung disease associated with increased lung cancer risk. Although previous studies have shown that IPF worsens the survival of patients with lung cancer, whether IPF independently affects cancer malignancy and prognosis remains inconclusive. Extracellular vesicles (EVs) have recently emerged as active carriers of molecular biomarkers and mediators of intercellular communication in lung homeostasis and pathogenesis. EV cargo-mediated fibroblast-tumor cell communication might participate in the development and progression of lung cancer by modulating various signaling pathways. In this study, we examined the impact of lung fibroblast (LF)-derived EVs on non-small cell lung cancer (NSCLC) malignancy in the IPF microenvironment. Here, we showed that LFs derived from patients with IPF have phenotypes of myofibroblast differentiation and cellular senescence. Furthermore, we found that IPF LF-derived EVs have markedly altered microRNA compositions and exert proproliferative functions on NSCLC cells. Mechanistically, the phenotype was attributed mainly to the enrichment of miR-19a in IPF LF-derived EVs. As a downstream signaling pathway, mir-19a in IPF LF-derived EVs regulates ZMYND11-mediated c-Myc activation in NSCLC, potentially contributing to the poor prognosis of patients with NSCLC with IPF. Our discoveries provide novel mechanistic insights for understanding lung cancer progression in the IPF microenvironment. Accordingly, blocking the secretion of IPF LF-derived EV miR-19a and their signaling pathways is a potential therapeutic strategy for managing IPF and lung cancer progression.

Inhibition of Nuclear Factor Kappa B Prevents the Development of Experimental Periapical Lesions.

INTRODUCTION: Nuclear factor kappa B (NF-κB) is an important transcriptional regulator of angiogenesis involving B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) signaling pathways. Thus, inhibition of NF-κB may suppress the development of periapical lesions via blockage of angiogenesis. Accordingly, we examined the effects of NF-κB decoy oligodeoxynucleotide (ODN) treatment on experimentally induced periapical lesions. METHODS: Periapical lesions were induced in the mandibular first molars of 5-week-old male Wistar rats by the application of lipopolysaccharide to the pulp. NF-κB decoy ODN or NF-κB decoy scramble (control) was injected intraperitoneally every 7 days, starting 1 day before pulp exposure. After 28 days, the samples were retrieved, and digital radiographs were taken for radiomorphometry. Samples were processed for (1) immunohistochemistry of CD31, Bcl-2, and Bax; (2) laser capture microdissection to analyze Bcl-2, Bax, chemokine (C-X-C motif) ligand 1 (CXCL1), CXC receptor 2 (CXCR2), and vascular endothelial cell growth factor receptor 2 (VEGFR2) messenger RNA (mRNA) expression in CD31+ endothelial cells; (3) enzyme-linked immunosorbent assay to determine NF-κB/p65 activity; and (4) Western blotting for vascular endothelial growth factor expression. RESULTS: NF-κB decoy ODN treatment significantly reduced lesion size, NF-κB/p65 activity, and the density of CD31+ endothelial cells in the lesion. NF-κB decoy ODNs also down-regulated CXCL1, CXCR2, and VEGFR2 mRNAs and up-regulated Bax mRNA in endothelial cells but did not affect Bcl2 mRNA in endothelial cells. Vascular endothelial growth factor protein expression in the lesions was significantly decreased. CONCLUSIONS: The inhibition of NF-κB activity by decoy ODN treatment suppressed the development of experimentally induced periapical lesions with a concomitant reduction in angiogenic responses in endothelial cells.

Implantation of Endothelial Cells with Mesenchymal Stem Cells Accelerates Dental Pulp Tissue Regeneration/Healing in Pulpotomized Rat Molars.

INTRODUCTION: This study aimed to examine whether the implantation of mesenchymal stem cells (MSCs) with endothelial cells (ECs) accelerates pulp tissue regeneration/healing and induces dentin bridge formation in a rat model of molar coronal pulp regeneration. METHODS: The maxillary first molars of Wistar rats were subjected to pulpotomy. Then, pulp chambers were implanted with biodegradable hydrogel-made scaffolds carrying MSCs together or without dermal microvascular ECs, and the cavities were sealed with mineral trioxide aggregate. After 14 days, pulp samples were analyzed by immunohistochemistry; messenger RNA expression of B-cell lymphoma 2 (Bcl-2), chemokine (C-X-C motif) ligand 1 (Cxcl1), CXC receptor 2 (Cxcr2), and dentin sialophosphoprotein (Dspp) by quantitative polymerase chain reaction, and protein expression of nestin and vascular endothelial growth factor by Western blotting. RESULTS: Teeth coimplanted with MSCs and ECs showed pulp healing with complete dentin bridge formation, whereas those implanted with MSCs alone had incomplete dentin bridges. Bcl-2, Cxcl1, Cxcr2, and Dspp messenger RNA levels were significantly up-regulated in the pulp of MSC/EC-implanted teeth compared with those in MSC-implanted teeth. Immunohistochemical analysis revealed the expression of nestin in odontoblastlike cells under dentin bridges in the MSC/EC coimplanted group. The density of CD31-expressing ECs and the expression of nestin and vascular endothelial growth factor proteins were significantly up-regulated in the MSC/EC-implanted pulp compared with the MSC-implanted pulp. CONCLUSIONS: The implantation of ECs with MSCs accelerated pulp tissue regeneration/healing and dentin bridge formation, up-regulated the expression of proangiogenic factors, and increased the density of ECs in pulpotomized rat molars.

Megalin Blockade with Cilastatin Suppresses Drug-Induced Nephrotoxicity.

Nephrotoxicity induced by antimicrobial or anticancer drugs is a serious clinical problem. Megalin, an endocytic receptor expressed at the apical membranes of proximal tubules, mediates the nephrotoxicity of aminoglycosides and colistin, key antimicrobials for multidrug-resistant organisms. The mechanisms underlying the nephrotoxicity induced by vancomycin, an antimicrobial for methicillin-resistant Staphylococcus aureus, and cisplatin, an important anticancer drug, are unknown, although the nephrotoxicity of these drugs and gentamicin, an aminoglycoside, is suppressed experimentally with cilastatin. In the clinical setting, cilastatin has been used safely to suppress dehydropeptidase-I-mediated renal metabolism of imipenem, a carbapenem antimicrobial, and thereby limit tubular injury. Here, we tested the hypothesis that cilastatin also blocks megalin-mediated uptake of vancomycin, cisplatin, colistin, and aminoglycosides, thereby limiting the nephrotoxicity of these drugs. Quartz crystal microbalance analysis showed that megalin also binds vancomycin and cisplatin and that cilastatin competes with megalin for binding to gentamicin, colistin, vancomycin, and cisplatin. In kidney-specific mosaic megalin knockout mice treated with colistin, vancomycin, or cisplatin, the megalin-replete proximal tubule epithelial cells exhibited signs of injury, whereas the megalin-deficient cells did not. Furthermore, concomitant cilastatin administration suppressed colistin-induced nephrotoxicity in C57BL/6J mice. Notably, cilastatin did not inhibit the antibacterial activity of gentamicin, colistin, or vancomycin in vitro, just as cilastatin did not affect the anticancer activity of cisplatin in previous studies. In conclusion, megalin blockade with cilastatin efficiently suppresses the nephrotoxicity induced by gentamicin, colistin, vancomycin, or cisplatin. Cilastatin may be a promising agent for inhibiting various forms of drug-induced nephrotoxicity mediated via megalin in the clinical setting.

Dental pulp tissue engineering of pulpotomized rat molars with bone marrow mesenchymal stem cells.

The major goal of dental pulp tissue engineering is to enable the healing of inflamed tissue or to replace necrotic pulp tissue with newly formed dental pulp tissue. Here, we report a protocol for pulp tissue engineering in vivo in pulpotomized rat teeth using constructs of rat bone marrow mesenchymal stem cells, preformed biodegradable scaffolds, and hydrogel. The constructs were implanted into pulpotomized pulp chambers for 3, 7, or 14 days. At 3 days, cells were located mainly along the preformed scaffolds. At 7 days, pulp tissue regeneration was observed in almost the entire implanted region. At 14 days, pulp tissue regeneration further progressed throughout the implanted region. In immunohistochemistry, at 3 days, a number of small and round macrophages immunoreactive to CD68 were predominantly distributed around the scaffolds. The density of CD68+ macrophages decreased until 14 days. On the other hand, nestin-expressing odontoblast-like cells beneath the dentin at the border of implanted region increased until 14 days. Quantitative gene expression analysis revealed that odontoblast differentiation marker dentin sialophosphoprotein mRNA in the implanted region gradually increased until 14 days. Together, the results suggested that regeneration of dental pulp tissue had occurred. Thus, our study provides a novel experimental rat model of dental pulp regeneration.

Megalin-Mediated Tubuloglomerular Alterations in High-Fat Diet-Induced Kidney Disease.

Obesity, an important risk factor for metabolic syndrome (MetS) and cardiovascular disease, is often complicated by CKD, which further increases cardiovascular risk and causes ESRD. To elucidate the mechanism underlying this relationship, we investigated the role of the endocytic receptor megalin in proximal tubule epithelial cells (PTECs). We studied a high-fat diet (HFD)-induced obesity/MetS model using kidney-specific mosaic megalin knockout (KO) mice. Compared with control littermates fed a normal-fat diet, control littermates fed an HFD for 12 weeks showed autolysosomal dysfunction with autophagy impairment and increased expression of hypertrophy, lipid peroxidation, and senescence markers in PTECs of the S2 segment, peritubular capillary rarefaction with localized interstitial fibrosis, and glomerular hypertrophy with mesangial expansion. These were ameliorated in HFD-fed megalin KO mice, even though these mice had the same levels of obesity, dyslipidemia, and hyperglycemia as HFD-fed control mice. Intravital renal imaging of HFD-fed wild-type mice also demonstrated the accumulation of autofluorescent lipofuscin-like substances in PTECs of the S2 segment, accompanied by focal narrowing of tubular lumens and peritubular capillaries. In cultured PTECs, fatty acid-rich albumin induced the increased expression of genes encoding PDGF-B and monocyte chemoattractant protein-1 via megalin, with large (auto)lysosome formation, compared with fatty acid-depleted albumin. Collectively, the megalin-mediated endocytic handling of glomerular-filtered (lipo)toxic substances appears to be involved primarily in hypertrophic and senescent PTEC injury with autophagy impairment, causing peritubular capillary damage and retrograde glomerular alterations in HFD-induced kidney disease. Megalin could be a therapeutic target for obesity/MetS-related CKD, independently of weight, dyslipidemia, and hyperglycemia modification.

Expression of angiogenic factors in rat periapical lesions.

INTRODUCTION: Angiogenic factors such as VEGFR2 (vascular endothelial cell growth factor receptor 2), Bcl-2 (a prosurvival and proangiogenic signaling molecule), and chemokine (C-X-C motif) ligand 1 (CXCL1) (a proangiogenic chemokine) may have critical roles in enhancing the establishment of apical periodontitis. To understand the role of these factors in the pathogenesis of apical periodontitis, we conducted immunohistochemical and molecular biological analysis. METHODS: Apical periodontitis was induced in the lower first molars of Wistar rats by making unsealed pulp exposures. After, 14, 21, and 28 days, the molars were retrieved, embedded as frozen sample blocks, and cut in a cryostat. Normal lower first molars served as controls. Immunostaining for CD31 (a marker for endothelial cells), Bcl-2, and real-time polymerase chain reaction analysis of VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA were performed. In the real-time polymerase chain reaction analysis, messenger RNA was extracted from CD31-stained endothelial cells that were retrieved with laser capture microdissection. For statistical analysis of immunohistochemistry, the immunostained area was plotted, and pixel counts were determined. Then, the percentage of the immunostained area in the total area was calculated. RESULTS: The density of the CD31-stained area increased until 21 days after pulp exposure. On the other hand, Bcl-2-stained area showed the highest density at 14 days (active lesion expanding phase) and then decreased until 28 days (lesion stability phase). VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells showed the highest levels at 14 days and then decreased until 28 days. CONCLUSIONS: The increase in microvascular density and the up-regulation of VEGFR2, Bcl-2, CXCL1, and CXCR2 messenger RNA expression in endothelial cells took place coincidently with the expanding phase of experimentally induced periapical lesions. These data suggest that these angiogenic factors play a role in the lesion development.

Rapid and transient upregulation of CCL11 (eotaxin-1) in mouse ovary during terminal stages of follicular development.

PROBLEM: This study aimed to investigate the regulation of expression, localization and physiological role of the CCL11/CCR3 axis in mouse ovary during the periovulatory period. METHOD OF STUDY: CCL11/CCR3 expression in the mouse ovary after treatment with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later was assessed in vivo and in 3-dimensional cultures in vitro. RESULTS: Real-time RT-PCR analyses revealed transient CCL11 mRNA upregulation 6 hr after hCG treatment. Immunohistochemical staining of serial ovarian sections demonstrated overlapping expression of CCL11, CCR3 and CD31 endothelial cell marker in the theca-interstitial layer at 10 hr after hCG treatment. In vitro 3-dimensional cultures of periovulatory ovarian tissues demonstrated that treatment with anti-CCL11 neutralizing antibody significantly decreased CD31 transcript. CONCLUSIONS: Gonadotropin surge leads to transient CCL11/CCR3 axis upregulation in the ovarian theca-interstitial layer, suggesting that it is involved in periovulatory physiological processes by affecting follicular vessels.

Gene expression analysis of resident macrophages in lipopolysaccharide-stimulated rat molar pulps.

INTRODUCTION: In normal dental pulp, a considerable number of resident macrophages are distributed. This study was designed to analyze the expression levels of genes associated with differentiation and function of resident macrophages in rat molar pulps stimulated with lipopolysaccharide (LPS). METHODS: Mandibular first molars of 7-week-old male Wistar rats were used. After transcardiac perfusion with a culture medium to preserve tissue integrity, pulpotomy and LPS application were carried out on the experimental teeth, and then dissected mandibles were subjected to whole-tooth culture for 3 days. Normal teeth and pulpotomized teeth without LPS served as controls. The specimens were then immunostained for ED1 (CD68, a general macrophage marker) and ED2 (CD163, a resident macrophage marker). Real-time polymerase chain reaction for Toll-like receptor 4 (TLR4), CD14, chemokine receptors (CCR2 and CX3CR1), and colony-stimulating factor-1 (CSF1) mRNAs was carried out after laser capture microdissection of ED1+ and ED2+ cells. RESULTS: LPS-treated pulps showed significant increases in (1) density of ED1+ and ED2+ cells beneath the amputation site and (2) expression levels of TLR4, CD14, CSF1, and CX3CR1 mRNAs, as compared with non-LPS-treated groups. CCR2 mRNA showed no significant difference between each group. CONCLUSIONS: LPS treatment of cultured rat molars caused the accumulation of resident macrophages and enhanced the expression of TLR4, CD14, CSF1, and CX3CR1 mRNAs in these cells. Up-regulation of these molecules might be involved in the differentiation and subsequent migration of resident macrophages of the pulp.

Laser-capture microdissection for Factor VIII-expressing endothelial cells in cancer tissues.

The immunostaining based Laser-capture microdissection (LCM) method called immune-LCM allows us to quantify the mRNA. Immune-LCM has recently been introduced to enhance identification of cells carrying a particular protein from frozen tissue samples. We have recently performed the immune-LCM of formaldehyde-fixated, paraffin-embedded tissues immunostained with a monoclonal antibody Factor VIII. This method could be useful for quantitative gene expression analysis in blood vessels from tumors of patients that have been treated with antiangiogenic drugs, allowing for validation of the effect of drug on the expected targets. Such capability might be exceedingly useful for the evaluation of the bioactivity of new drugs. This method is also useful to compare gene expression patterns in tumor cells versus endothelial cells during tumor progression or tumor angiogenesis.

Megalin is downregulated via LPS-TNF-α-ERK1/2 signaling pathway in proximal tubule cells.

Expression and function of megalin, an endocytic receptor in proximal tubule cells (PTCs), are reduced in diabetic nephropathy, involved in the development of proteinuria/albuminuria. Lipopolysaccharide (LPS) is chronically increased in diabetic sera, by the mechanism called metabolic endotoxemia. We investigated low-level LPS-mediated signaling that regulates megalin expression in immortalized rat PTCs (IRPTCs). Incubation of the cells with LPS (10 ng/ml) for 48 h suppressed megalin protein expression and its endocytic function. TNF-α mRNA expression was increased by LPS treatment, and knockdown of the mRNA with siRNA inhibited LPS-mediated downregulation of megalin mRNA expression at the 24-h time point. Incubation of IRPTCs with exogenous TNF-α also suppressed megalin mRNA and protein expression at the 24- and 48-h time points, respectively. MEK1 inhibitor PD98059 competed partially but significantly TNF-α-mediated downregulation of megalin mRNA expression. Collectively, low-level LPS-mediated TNF-α-ERK1/2 signaling pathway is involved in downregulation of megalin expression in IRPTCs.

The role of N-methyl-D-aspartate receptor subunits in the rat thalamic mediodorsal nucleus during central sensitization.

We have identified tooth pulp-driven neurons (TPDNs) in the thalamic mediodorsal nucleus (MD) in rats and showed that the TPDNs' responsiveness in the MD is increased by chemical conditioning stimulation of allyl-isothiocyanate (mustard oil) to the molar tooth pulp. The aim of the present study was to address the role of N-methyl-d-aspartate receptors (NMDA receptors) in the sensitized central nervous system following the mustard oil application to the rat tooth pulp. Microinjection of MK-801, a noncompetitive NMDA receptor antagonist, to the thalamic MD nucleus reduced the TPDNs' responsiveness in the thalamic MD nucleus. Gene expression analysis showed that expression levels of NMDA receptor subunits NR2A and NR2D mRNAs in the thalamus were increased by the mustard oil application and that the increases were reduced by MK-801. When naloxone, an opioid receptor antagonist, was given systemically following the MK801 microinjection, the TPDNs' responsiveness was rekindled and expression levels of NR2D and NR2A mRNAs were increased. Moreover, lidocaine pretreatment abolished the mustard oil-induced upregulation of NR2D and NR2A mRNAs. These results suggest that, during central sensitization, interaction of NMDA receptors and endogeneous opioid-related inhibitory mechanisms plays critical role in the alteration of the TPDNs' responsiveness in the thalamic MD nucleus.

Increased gene expression of Toll-like receptors and antigen-presenting cell-related molecules in the onset of experimentally induced furcation lesions of endodontic origin in rat molars.

INTRODUCTION: Early immunopathogenic mechanisms behind pulp infection-induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection-induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. METHODS: Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. RESULTS: Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. CONCLUSIONS: These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation.

Artificial dental pulp exposure injury up-regulates antigen-presenting cell-related molecules in rat central nervous system.

INTRODUCTION: Bacterial infection and resulting inflammation of the dental pulp might not only trigger neuroimmune interactions in this tissue but also sensitize the central nervous system (CNS) such as the thalamus via nociceptive neurons. Thus, immunopathologic changes in the rat thalamus that take place after pulp inflammation were investigated. METHODS: Pulp exposure was made in mandibular right first molars of 5-week-old Wistar rats. After 24 hours, the thalamus was retrieved and subjected to either immunohistochemistry for class II major histocompatibility complex (MHC) molecules and glial fibrillary acidic protein (GFAP) or mRNA expression analysis of antigen-presenting cell-related molecules and N-methyl-D-aspartate receptor 2D subunit (NR2D) by means of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: At 24 hours after pulp exposure, the density of class II MHC molecule-expressing and GFAP-expressing cells was increased in the contralateral thalamus. Gene expression analysis revealed the up-regulation of class II MHC molecules, CD80, CD83, CD86, and NR2D in the contralateral thalamus, as compared with the ipsilateral thalamus. CONCLUSIONS: These results suggest the signal of pulp inflammation induces neuronal activation in the CNS.

Laser capture microdissection in dentistry.

Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry.

Gene expression analysis of acutely traumatized pulps.

INTRODUCTION: Vertical root fracture of vital teeth is a relatively rare occurrence. To address early molecular biologic events that take place in the pulp of such cases, we measured mRNA expression levels of selected molecules related to nociception, bacterial pattern recognition, and hard tissue repair/mineralization. METHODS: Three extracted roots obtained from vital molars diagnosed as vertical root fracture were used. The samples were first analyzed with light and transmission electron microscopy. Then mRNA expression in the apical (root fractured) and coronal portions of the pulp was analyzed by using reverse transcription-polymerase chain reaction (PCR) or real-time PCR after laser capture microdissection. RESULTS: In all the samples, cracks and vital pulp tissue, but not signs of infection and inflammation, were recognized in the apical portion of the fractured root. The gene expression analysis showed that mRNAs of pattern recognition receptors (CD14, Toll-like receptor 2, and Toll-like receptor 4) and neurokinin-1 receptor were equally expressed in both regions of the pulp. On the other hand, mRNA expression levels of hard tissue-associated proteins (osteopontin, osteonectin, and osteocalcin) and calcium channel voltage-dependent alpha 2 delta subunit 1 (CACNA2D1) in the apical portion of the pulp tissue and periodontal ligaments were significantly up-regulated, as compared with those in the coronal portion of the pulp. CONCLUSIONS: In the traumatized apical pulp of vertically root-fractured vital teeth, elevated mRNA expression of CACNA2D1, a nociception-related molecule, and proteins related to hard tissue repair/mineralization occurs under noninfectious condition.

Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

Characteristics of resident dendritic cells in various regions of rat periodontal ligament.

In the periodontal ligament (PDL) of normal rat molars, a considerable number of dendritic cells (DCs) are distributed as resident immunocompetent cells. We have hypothesized that the resident type of DCs can alter their morphology and phenotype to adapt to the microenvironment of the PDL. Thus, we have compared the characteristics of DCs in various regions of rat PDL by immunoelectron microscopy and gene expression analysis by means of reverse transcription/polymerase chain reaction. DCs in the periapical region show immature characteristics, i.e., small round profiles and few organelles. In the mesial region, which is the most stable area in the PDL, the density of DCs is the highest. DCs in the furcal region show the characteristics of activated antigen-presenting cells, i.e., CD86(high) expression, electron-dense lysosomes, and cell-to-cell contacts with lymphocytes. When the PDL is bacterially challenged by pulp exposure, the number of CD86+ DCs and contacts with lymphocytes increase in the furcal region. Gene expression analysis for the furcal region has also shown the up-regulation of the major histocompatibility complex class II alpha-chain, CD83, CD86, and the antigen-presenting cell-lectin-like receptor A1. Thus, we have demonstrated heterogeneity in resident DCs. This may be associated with the difference in DCs regarding their state of maturation/activation, which may further reflect microenvironmental differences such as the amount of external antigens.