Combination of plasma MMPs and PD-1-binding soluble PD-L1 predicts recurrence in gastric cancer and the efficacy of immune checkpoint inhibitors in non-small cell lung cancer.

Background: The tumor microenvironment (TME) impacts the therapeutic efficacy of immune checkpoint inhibitors (ICIs). No liquid biomarkers are available to evaluate TME heterogeneity. Here, we investigated the clinical significance of PD-1-binding soluble PD-L1 (bsPD-L1) in gastric cancer (GC) patients and non-small cell lung cancer (NSCLC) patients treated with PD-1/PD-L1 blockade. Methods: We examined bsPD-L1, matrix metalloproteinases (MMPs), and IFN-γ levels in plasma samples from GC patients (n = 117) prior to surgery and NSCLC patients (n = 72) prior to and 2 months after ICI treatment. We also examined extracellular matrix (ECM) integrity, PD-L1 expression, and T cell infiltration in tumor tissues from 25 GC patients by Elastica Masson-Goldner staining and immunohistochemical staining for PD-L1 and CD3, respectively. Results: bsPD-L1 was detected in 17/117 GC patients and 16/72 NSCLC patients. bsPD-L1 showed strong or moderate correlations with plasma MMP13 or MMP3 levels, respectively, in both GC and NSCLC patients. bsPD-L1 expression in GC was associated with IFN-γ levels and intra-tumoral T cell infiltration, whereas MMP13 levels were associated with loss of ECM integrity, allowing tumor cells to access blood vessels. Plasma MMP3 and MMP13 levels were altered during ICI treatment. Combined bsPD-L1 and MMP status had higher predictive accuracy to identify two patient groups with favorable and poor prognosis than tumor PD-L1 expression: bsPD-L1+MMP13high in GC and bsPD-L1+(MMP3 and MMP13)increased in NSCLC were associated with poor prognosis, whereas bsPD-L1+MMP13low in GC and bsPD-L1+(MMP3 or MMP13)decreased in NSCLC were associated with favorable prognosis. Conclusion: Plasma bsPD-L1 and MMP13 levels indicate T cell response and loss of ECM integrity, respectively, in the TME. The combination of bsPD-L1 and MMPs may represent a non-invasive tool to predict recurrence in GC and the efficacy of ICIs in NSCLC.

High Phase-Purity and Composition-Tunable Ferromagnetic Icosahedral Quasicrystal.

We discovered a ferromagnetic Au-Ga-Dy icosahedral quasicrystal (i QC), not only with high phase purity but also with tunable composition. The isothermal magnetization of the polycrystalline ferromagnetic i QC was closely investigated and the mean-field-like nature of the ferromagnetic transition is elucidated. Moreover, the maximum Weiss temperature (θ_{p}) of the i QCs was found at the electrons-per-atom (e/a) ratio of 1.70 being well consistent with those of ACs, validating tunability of the magnetic properties of i QCs on the basis of θ_{p}-e/a scheme for the first time. Thus, the present work provided direct evidence that the magnetism of the i QCs depends on the e/a ratio or the Fermi energy, paving the way for future studies on various exotic magnetic textures formed on a quasiperiodic lattice through the e/a ratio.

Experimental Observation of Long-Range Magnetic Order in Icosahedral Quasicrystals.

Quasicrystals (QCs), first discovered in 1984, generally do not exhibit long-range magnetic order. Here, we report on long-range magnetic order in the real icosahedral quasicrystals (i QCs) Au-Ga-Gd and Au-Ga-Tb. The Au65Ga20Gd15 i QC exhibits a ferromagnetic transition at TC = 23 K, manifested as a sharp anomaly in both magnetic susceptibility and specific heat measurements, along with an appearance of magnetic Bragg peak below TC. This is the first observation of long-range magnetic order in a real quasicrystal, in contrast to the spin-glass-like behaviors observed for the other magnetic quasicrystals found to date. Moreover, when Gd is replaced by Tb, i.e., for the Au65Ga20Tb15 i QC, a ferromagnetic behavior is still retained with TC = 16 K. Although the sharp anomaly in the specific heat observed for the Au65Ga20Gd15 i QC becomes broadened upon Tb substitution, neutron diffraction experiments clearly show marked development of magnetic Bragg peaks just below TC, indicating long-range magnetic order for the Au65Ga20Tb15 i QC also. Our findings can contribute to the further investigation of exotic magnetic orders formed on real quasiperiodic lattices with unprecedented highest global symmetry, i.e., icosahedral symmetry.

Toll­like receptor 4 plays a tumor­suppressive role in cutaneous squamous cell carcinoma.

Toll­like receptor 4 (TLR4), a key regulator of the innate immune system, is expressed not only in immune cells, but also in a number of cancer cells. A biological role for TLR4 in cutaneous squamous cell carcinoma (SCC), however, is unclear. In this study, we first examined TLR4 expression and localization in cases of SCC, actinic keratosis (AK) and Bowen's disease (BD) by immunohistochemistry. TLR4 expression was significantly higher in the SCC than in the AK or BD tissues. We then determined the TLR4 expression level in vivo, in 3 histological subtypes of SCC. TLR4 expression in poorly differentiated SCC was significantly lower compared with that of the moderately and well­differentiated type. In addition, the CD44 immunoreactivity tended to be high in the cell membrane of poorly differentiated SCC. Of note, poorly differentiated SCC is a risk factor of unfavorable outcomes in affected patients. We then assessed the biological role of TLR4 in HSC­1 and HSC­5 SCC cells and HaCaT human keratinocytes. TLR4 knockdown by transfection with siRNA accelerated HSC­1 and HaCaT cell migration and invasion compared to the control siRNA­transfected cells. TLR4 knockdown resulted in an increased CD44 expression and in an enhanced filopodia protrusion formation, particularly in HSC­1. On the whole, these results suggest that a reduced TLR4 expression enhances the malignant features in SCC cases and cultured SCC cell lines. TLR4 may thus play an anti­tumor role in cutaneous SCC.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells.

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.

Higher expression of EpCAM is associated with poor clinical and pathological responses in breast cancer patients undergoing neoadjuvant chemotherapy.

Neoadjuvant chemotherapy (NAC) is a standard regimen in treatment of breast cancer patients, but some are resistant to NAC. We hypothesized that breast cancer cells overexpressing epithelial cell adhesion molecule (EpCAM) could be resistant to NAC, contributing to a poor prognosis. Seventy patients with breast cancer were treated with NAC. Core needle biopsy (CNB) specimens and resected tumors before and after NAC, respectively, were examined for expression of EpCAM. In resected tumors, high EpCAM expression correlated with lymphovascular invasion status and nuclear grade (P = 0.01 and 0.008, respectively), and was associated with poor pathological and clinical responses (P < 0.001). High tumoral EpCAM expression in resected tumor was independently related to a poor pathological response. Patients with high EpCAM expression before and after NAC (high-to-high group) showed worse pathological and clinical responses (P = 0.008 and <0.001, respectively) than the patients with high and low EpCAM expression before and after NAC, respectively (high-to-low group). The overall survival rate of the high-to-high group appeared shorter compared with the high-to low-group (P = 0.049). Our findings imply that higher levels of EpCAM in breast cancer may be associated with poor response to NAC via a potential chemoresistant effect.

Insulin-like growth factor 2 mRNA-binding protein-3 as a marker for distinguishing between cutaneous squamous cell carcinoma and keratoacanthoma.

In the histopathological diagnosis of cutaneous tumors, the differential diagnosis of squamous cell carcinoma (SCC) with crateriform architecture and keratoacanthoma (KA) is often difficult so an accurate understanding of the biological features and the identification of reliable markers of SCC and KA are crucial issues. Insulin-like growth factor 2 mRNA-binding protein-3 (IGF2BP3, also known as IMP3) is thought of as a bona fide oncofetal protein, which is overexpressed and is involved in cell proliferation, migration, and invasion in several kinds of tumors. However, the role of IMP3 in cutaneous SCC and KA has not been well studied. Therefore, we focused on studying the biological functions of IMP3 in SCC and KA. In human skin SCC cell lines, HSC-1 and HSC-5, and the human keratinocyte cell line, HaCaT, IMP3 mRNA levels were significantly higher than that of normal human skin. The knockdown of IMP3 expression reduced the proliferation of HSC-1, and significantly reduced invasion by HSC-1 and HSC-5. In contrast, the knockdown of IMP3 did not significantly affect invasion by HaCaT cells. In immunohistochemical studies of SCC and KA tissues, the Ki-67 labeling index (LI) of the suprabasal cell layer was significantly higher in SCC, compared with KA tissues and the tumor-free margin (TFM) adjacent to SCC and KA. Most SCC tissues stained strongly positive for IMP3, but KA tissues and TFM were mostly negative for IMP3. The Ki-67 LI of the IMP3-positive group was significantly higher than that of the IMP3-negative group in the suprabasal cell layer of SCC. These results suggest that IMP3 plays an important role in proliferation and, more significantly, in the invasion of SCC, and may be a suitable marker for the histopathological diagnosis of SCC with a crateriform architecture and KA. Furthermore, IMP3 may potentially be a new therapeutic target for SCC.

Modified six-minute walk test: number of steps per second.

The 6-min walk test (6MWT) has been used to examine subjective dyspnea, predict mortality and measure clinical outcomes in studies of patients with chronic pulmonary or heart disease. Although the 6MWT is useful to assess the general ability to perform daily physical activity, it is difficult to evaluate time-dependent responses. To improve the 6MWT, we devised a new index, which is the number of steps walked per second (NSPS). We performed the 6MWT in 11 healthy subjects and 7 patients with chronic obstructive pulmonary disease (COPD) and calculated the NSPS. The mean NSPS was significantly higher in the healthy subjects than in the COPD patients, while the coefficient of variation of the NSPS was significantly smaller in healthy subjects compared with COPD patients. Calculation of the NSPS was useful to evaluate the walking pattern. This modified 6MWT may be helpful for assessing the efficacy of rehabilitation and drug therapy for COPD.

Altered expression of fibroblast growth factor receptor 2 isoform IIIc: relevance to endometrioid adenocarcinoma carcinogenesis and histological differentiation.

Fibroblast growth factor receptor 2 (FGFR2) is activated in many cancers and considered as a potential therapeutic molecular target including for endometrial endometrioid carcinoma (EEC). Overexpression of FGFR2 isoform IIIc (FGFR2IIIc) has been shown to be associated with carcinogenesis in various cancers, but its expression in EEC has not been reported yet to the best of our knowledge. In this study, we identified roles for FGFR2IIIc in EEC carcinogenesis and demonstrated its diagnostic and prognostic values in EEC. FGFR2IIIc expression was compared between 10 normal endometrium, 10 atypical endometrial hyperplasias, and 47 EEC specimens using immunohistochemistry and quantitative real-time PCR. Atypical hyperplasia, Grade 1 (G1), and Grade 2 (G2) differentiated EEC tissues showed significantly higher FGFR2IIIc expression than normal endometrium tissue. However, as compared to G1 and G2 EECs, Grade 3 (G3) differentiated EEC tissue showed lower FGFR2IIIc expression (P<0.05). There was no significant association between FGFR2IIIc expression and patient age, lymph node metastasis, and EEC stage. These results suggest that altered FGFR2IIIc expression plays an important role in EEC carcinogenesis and may occur in precancerous tissues. However, FGFR2IIIc appears to be not related to EEC progression. Some G3 EECs may develop through different carcinogenic processes than G1 and G2 EECs.

Nodular fasciitis in the parotid gland: a case report and review of the literature.

Nodular fasciitis (NF) is a benign, reactive lesion with a self-limiting process. Because NF is rare in the parotid gland and has many cytological similarities to other benign or malignant tumors, cytological misinterpretation is common. The patient, a 30-year-old woman, had a painless mass in her right parotid gland. Fine needle aspiration cytology (FNAC) was performed. Spindle cells with basophilic and well-demarcated cytoplasm were observed in a mucoid-like background. The mucoid-like substance was metachromatic and appeared to be the matrix of PA. Histopathologically, spindle-shaped cells with intervening birefringent mature collagen were arranged in short irregular bundles. Prominent mucoid-like matrixes as well as few infiltrating neutrophils and lymphocytes were found in the background. Lesional cells were positive for CD10 and ß-catenin in the cytoplasm, but negative for cytokeratin, the S-100 protein, CD34, and neurofilament. Ultimately, this patient was diagnosed with NF. In FNAC of the parotid gland region, distinguishing NF from other real tumors is important for deciding treatment strategies.

Comparison of fixation methods for preservation of morphology, RNAs, and proteins from paraffin-embedded human cancer cell-implanted mouse models.

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Keratinocyte growth factor induces vascular endothelial growth factor-A expression in colorectal cancer cells.

Keratinocyte growth factor (KGF), which is also called fibroblast growth factor (FGF)-7, belongs to the FGF family. KGF is not commonly produced by human cancer cells, but the KGF receptor (KGFR) is expressed in most cancer cells and particularly highly expressed in well-differentiated types of cancer. Recently, it has been reported that vascular endothelial growth factor (VEGF)-A expression is induced by KGF in pancreatic cancer cells. VEGF-A is produced by some cancer cells and plays important roles in the angiogenesis and metastasis of cancer cells including those in the colorectum. In this study, we examined whether recombinant human KGF (rhKGF) induces major angiogenic growth factors including VEGF-A, FGF-2 and hepatocyte growth factor (HGF) in human colorectal cancer cells (HCT-15), which express a high level of KGFR, but a low or negligible level of KGF. rhKGF significantly increased the VEGF-A expression level in a serum-free medium of HCT-15 cells, but FGF-2 and HGF expression levels were too low to detect. Furthermore, the expression levels of the angiogenic growth factors were evaluated in KGF-transfected HCT-15 cells, which were induced to stably overexpress KGF by KGF gene transfection and mock-transfected cells (Mock). KGF and VEGF-A expression levels in the cells and the protein concentrations in serum-free medium were significantly higher in KGF-transfected HCT-15 cells than in Mock cells. In contrast, the FGF-2 and HGF mRNA expression levels were not significantly different between KGF-transfected HCT-15 cells and Mock cells and the protein concentrations in serum-free medium of the cells were below the detection level. These findings suggest that administration of rhKGF and over-expression of endogenous KGF genes in colorectal cancer cells increase VEGF-A production and may relate to angiogenesis in cancer.

Expression and roles of keratinocyte growth factor and its receptor in esophageal cancer cells.

The keratinocyte growth factor receptor (KGFR), also known as FGFR2 IIIb, is mainly localized in epithelial cells and is activated by the keratinocyte growth factor (KGF) that is predominantly synthesized by mesenchymal cells. In this study, we examined the roles of KGFR and KGF in human esophageal cancer (EC). In noncancerous esophageal tissues, KGFR was localized in epithelial cells from the basal region of the epithelium to the lower one-third of the epithelium, and KGF was weakly localized in the basal to parabasal epithelial cells. On the other hand, Ki-67 was localized in the parabasal cells. In EC tissues, KGFR and KGF were expressed in cancer cells in 22 and 37 of 54 patients, respectively. The coexpression of KGFR and KGF in cancer cells was detected in 14 of 54 (26%) patients. Clinicopathologically, KGFR expression correlated with the well-differentiated cell type of EC (p<0.001), and KGF expression correlated with lymphatic invasion and lymph node metastasis (p=0.004 and 0.021, respectively). The coexpression of KGFR and KGF in cancer cells correlated with the well-differentiated cell type of EC (p=0.001). KGFR-positive, KGF-positive and KGFR/KGF coexpression patients tended to have shorter survival rates, but the survival rates were not statistically significantly different (p=0.44, 0.059 and 0.112, respectively). In human EC cell lines (TE-1, TE-8 and TE-11), KGFR mRNA was expressed but no KGF mRNA was detected. The KGFR mRNA level was highest in TE-1 cells, derived from well-differentiated SCC and lowest in TE-8 cells. KGFR was detected in the cancer cell lines by Western blot analysis. Recombinant human KGF significantly stimulated the growth of TE-8 and -11 cells, derived from moderately differentiated SCC, but had no effect on TE-1 cell growth. These results suggest that KGFR expression correlates with the differentiation of a normal esophageal epithelium and the well-differentiated cell type of EC. On the other hand, KGF may induce the growth of some EC cells in a paracrine manner and closely correlates with lymphatic invasion and lymph node metastasis.

Keratinocyte growth factor-transfection-stimulated adhesion of colorectal cancer cells to extracellular matrices.

The keratinocyte growth factor (KGF) regulates cell growth and behavior in an autocrine or paracrine manner. In colorectal cancer tissues, KGF is expressed in tumor cells and adjacent stromal fibroblasts. We have constructed a KGF-gene-transfected cell line (HCT15-KGF) from a colorectal cancer cell line, HCT-15, that expresses the KGF receptor, and studied the effects of KGF on cell behavior, particularly growth and adhesion to extracellular matrices (ECMs). The amount of KGF secreted from HCT15-KGF was significantly higher than that from a mock-transfected cell line (HCT15-MOCK). The modes of growth of these cell lines were similar. The degree of adhesion of HCT15-KGF to ECMs, including type-IV collagen and fibronectin was higher than that of HCT15-MOCK. The expressions of integrins in both cell lines were not significantly different. However, extracellular-regulated kinase-1 and -2 (ERK1/2) phosphorylation and focal adhesion kinase (FAK) expression that regulate the adhesive functions of integrin families were enhanced in HCT15-KGF. U0126, an inhibitor of the ERK upstream regulator MEK, attenuated the adhesion and spreading of HCT15-KGF cells to type-IV collagen. These results indicate that KGF enhances the adhesion of colorectal cancer cells to type-IV collagen through ERK and FAK signaling pathways.

Defined localization of nestin-expressing cells in L-arginine-induced acute pancreatitis.

OBJECTIVE: Nestin is a stem cell marker originally described as an intermediate filament protein expressed in neuroepithelial stem cells. In the pancreas, a small number of nestin-expressing cells, which are believed to represent either stem cells or progenitor cells, are known to be present in islets, as well as in some stellate cells, pericytes, and endothelial cells. We monitored pancreatic nestin expression to delineate the location of stem cells/progenitor cells in the pancreas after L-arginine-induced pancreatitis. METHODS: Male Wistar rats received 2 intraperitoneal injections of L-arginine, each consisting of 250 mg/100 g of body weight, and were killed 3, 6, and 12 hours and 1, 4, 7, and 14 days later. RESULTS: Serum amylase and lipase levels increased after L-arginine injection, maximal levels occurring at 3 and 12 hours postinjection, respectively. Six hours after L-arginine injection, interstitial edema was observed in the pancreas, whereas on day 4 postinjection, there was severe pancreatic necrosis. Neovascularization and ductal-ductular proliferation were also present in the pancreas. Immunohistochemical analysis revealed increased Ki-67 labeling in acinar cells and capillary endothelial cells. Immunoblotting using antinestin antibody revealed increased nestin expression after L-arginine injection. In the control rat pancreas, nestin immunoreactivity was detected in a few capillary endothelial cells in some islets. After L-arginine injection, nestin was expressed in proliferating capillary endothelial cells, in stellate cells surrounding ductular structures and in submesothelial cells. CONCLUSIONS: Transient nestin expression occurs in specific cell types during the proliferative stage after recovery from L-arginine-induced pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the regenerative capacity of the pancreas.

Effect of morpholino antisense oligonucleotide against lumican mRNA in human embryonic kidney (HEK) 293 cells.

Lumican is a member of the small-leucine-rich proteoglycan (SLRP) family and is overexpressed during wound healing of the cornea, in ischemic and reperfused heart, and in several cancer tissues. Lumican is considered to regulate the collagen fibril diameter and interfibrillar spacing. However, the effect of lumican on cell growth has not been adequately examined. In the present study, we attempted to clarify whether lumican contributes to human embryonic kidney (HEK) 293 cell growth, using the morpholino antisense oligonucleotide (m-anti oligo) against lumican mRNA. M-anti oligo is a novel oligonucleotide and exhibits a higher antisense activity, higher water solubility, and greater resistance to nucleases in target cells than phosphorothioate types of oligonucleotide. After delivery of m-anti oligo against lumican mRNA, the fluorescein 5-isothiocyanate (FITC) conjugated oligonucleotides were observed in the cytoplasm and nucleus of HEK 293 cells at 24 h by confocal laser microscopy. M-anti oligo for lumican mRNA strongly inhibited the synthesis of lumican protein in the HEK 293 cells, and the HEK cell growth rate was higher than those in the control groups. These findings may indicate that lumican protein has an inhibitory effect on HEK 293 cell growth in vitro.

Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in vascular smooth muscle cells.

Keratinocyte growth factor receptor (KGFR), also known as fibroblast growth factor receptor (FGFR)2 IIIb, is located in many types of epithelial cells and is activated by four known ligands (FGF-1, FGF-3, FGF-7 (also known as KGF) and FGF-10) that are predominantly synthesized by mesenchymal cells. In the early stage of atherosclerosis, vascular smooth muscle cells (VSMC) transform from a contractile to a synthetic phenotype, proliferate and migrate into the intima. Previously, FGF-7 mRNA expression was reported in VSMC, but KGFR mRNA was not detected. In the present study, we attempted to determine whether KGFR is localized in VSMC cultured from rat aorta and VSMC in human normal and atherosclerotic coronary arteries. Expression of KGFR mRNA and its protein was detected in cultured rat VSMC by reverse transcription-polymerase chain reaction and western blot analysis, respectively. Immunohistochemically, KGFR was localized in the VSMC of the outer layer of the media in normal human coronary arteries. Furthermore, it was localized in the VSMC of the media and thickened intima of atherosclerotic arteries. Recombinant FGF-7 and/or FGF-10 proteins stimulated the growth of cultured rat VSMC. These findings indicate that KGFR localized in VSMC may contribute to the proliferation of VSMC in normal and atherosclerotic arteries.

Transient and ectopic expression of lumican by acinar cells in L-arginine-induced acute pancreatitis.

Lumican is a member of a small leucine-rich proteoglycan family. We previously found that lumican mRNA and its protein were ectopically and highly expressed in acinar cells in chronic pancreatitis (CP)-like lesions close to pancreatic cancer cells. CP-like lesions are characterized by acinar and ductal-ductular cell proliferation with expanding fibrosis. This finding suggests that lumican is ectopically synthesized by acinar cells under chronic inflammatory conditions and plays a role in fibrosis of the pancreas. However, the expression and role of lumican in acute inflammatory changes of the pancreas are not completely elucidated. In the present study, we aim to clarify whether lumican mRNA and its protein are expressed in exocrine or endocrine components in acute pancreatitis (AP). For experimental AP, Wistar rats received an intraperitoneal injection of L-arginine. Western blot analysis showed an intense 50-kDa band corresponding to the lumican protein in normal and L-arginine-treated rat pancreas. After L-arginine injection, three intense bands at 42, 57, and 92 kDa were detected on day 1. Immunohistochemically, the lumican protein was localized in ductal and a few centroacinar cells in the normal pancreas. After L-arginine injection, an immature fibrosis with fragmented and loose collagen fibers was observed in AP on day 4 and lumican immunoreactivity was detected in the collagen fibers. Lumican mRNA was faintly detected in islet cells in the normal pancreas, but it was strongly expressed in acinar and islet cells on day 1. Furthermore, lumican mRNA was expressed in many proliferating fibroblasts on day 4 by in situ hybridization. These findings indicate that lumican is transiently synthesized by acinar cells and fibroblasts in AP. Lumican proteins synthesized by acinar cells, islet cells, and fibroblasts may contribute to immature and transient fibrosis of AP.

Floppy aortic valves without aortic root dilatation: clinical, histologic, and ultrastructural studies.

Gross anatomic, histologic and ultrastructural studies were made on 32 floppy aortic valves (FAVs) resected at the time of aortic valvular replacement for aortic regurgitation. Patients with the FAVs had relatively long clinical courses and had severe aortic regurgitation with mild symptoms of heart failure. The sizes of the mechanical valves implanted in the patients with FAVs were not large, indicating that the aortic regurgitation in these patients was not worsened by dilatation of the aortic ring. Two types of FAVs were recognized grossly, according to whether they showed abnormal cuspal thickening or thinning. Accumulations of myxoid material in the spongiosa were found in all FAVs, regardless of cuspal gross morphology. Histologically, the collagen fibers were sparse and irregularly arranged and elastic fibers were disrupted and finely granular in the myxomaotus areas of FAVs. Ultrastructurally, the myxomatous material consisted of numerous star-shaped proteoglycan granules associated with spiraling collagen fibrils and abnormal elastic fibers. Numerous spiraling collagen fibrils were observed especially at the border area of myxomatous change that extended from the spongiosa into the fibrosa. Abnormal elastic fibers had either a granular appearance of their amorphous components without microfibrils, or irregularly arranged masses of microfibrils without amorphous components. These abnormalities of connective tissue components, resulting from defective formation and/or increased degradation were similar to those in floppy mitral valves, and were related to the floppiness of cardiac valves.