Cytotoxic effects of the cigarette smoke extract of heated tobacco products on human oral squamous cell carcinoma: the role of reactive oxygen species and CaMKK2.

BACKGROUND: The increasing prevalence of heated tobacco products (HTPs) has heightened concerns regarding their potential health risks. Previous studies have demonstrated the toxicity of cigarette smoke extract (CSE) from traditional tobacco's mainstream smoke, even after the removal of nicotine and tar. Our study aimed to investigate the cytotoxicity of CSE derived from HTPs and traditional tobacco, with a particular focus on the role of reactive oxygen species (ROS) and intracellular Ca2+. METHODS: A human oral squamous cell carcinoma (OSCC) cell line, HSC-3 was utilized. To prepare CSE, aerosols from HTPs (IQOS) and traditional tobacco products (1R6F reference cigarette) were collected into cell culture media. A cell viability assay, apoptosis assay, western blotting, and Fluo-4 assay were conducted. Changes in ROS levels were measured using electron spin resonance spectroscopy and the high-sensitivity 2',7'-dichlorofluorescein diacetate assay. We performed a knockdown of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) by shRNA lentivirus in OSCC cells. RESULTS: CSE from both HTPs and traditional tobacco exhibited cytotoxic effects in OSCC cells. Exposure to CSE from both sources led to an increase in intracellular Ca2+ concentration and induced p38 phosphorylation. Additionally, these extracts prompted cell apoptosis and heightened ROS levels. N-acetylcysteine (NAC) mitigated the cytotoxic effects and p38 phosphorylation. Furthermore, the knockdown of CaMKK2 in HSC-3 cells reduced cytotoxicity, ROS production, and p38 phosphorylation in response to CSE. CONCLUSION: Our findings suggest that the CSE from both HTPs and traditional tobacco induce cytotoxicity. This toxicity is mediated by ROS, which are regulated through Ca2+ signaling and CaMKK2 pathways.

Alternative magnetic field exposure suppresses tumor growth via metabolic reprogramming.

Application of physical forces, ranging from ultrasound to electric fields, is recommended in various clinical practice guidelines, including those for treating cancers and bone fractures. However, the mechanistic details of such treatments are often inadequately understood, primarily due to the absence of comprehensive study models. In this study, we demonstrate that an alternating magnetic field (AMF) inherently possesses a direct anti-cancer effect by enhancing oxidative phosphorylation (OXPHOS) and thereby inducing metabolic reprogramming. We observed that the proliferation of human glioblastoma multiforme (GBM) cells (U87 and LN229) was inhibited upon exposure to AMF within a specific narrow frequency range, including around 227 kHz. In contrast, this exposure did not affect normal human astrocytes (NHA). Additionally, in mouse models implanted with human GBM cells in the brain, daily exposure to AMF for 30 min over 21 days significantly suppressed tumor growth and prolonged overall survival. This effect was associated with heightened reactive oxygen species (ROS) production and increased manganese superoxide dismutase (MnSOD) expression. The anti-cancer efficacy of AMF was diminished by either a mitochondrial complex IV inhibitor or a ROS scavenger. Along with these observations, there was a decrease in the extracellular acidification rate (ECAR) and an increase in the oxygen consumption rate (OCR). This suggests that AMF-induced metabolic reprogramming occurs in GBM cells but not in normal cells. Our results suggest that AMF exposure may offer a straightforward strategy to inhibit cancer cell growth by leveraging oxidative stress through metabolic reprogramming.

A case of delayed bleeding of the chest wall after VATS treated with transcatheter arterial embolization.

We report a case of delayed bleeding after video-assisted thoracic surgery (VATS) that was successfully treated with transcatheter arterial embolization. An 81-year-old woman underwent a pleural biopsy via VATS for pleural dissemination of lung cancer. The postoperative course was good, but 8 days later she was hospitalized for swelling in the right axilla and was admitted to our hospital with a diagnosis of delayed postoperative hemorrhage. Gauze compression was performed, and the patient was discharged without exacerbation of hematoma. However, 4 days later, she was hospitalized for rapidly worsening swelling and pain. Chest computed tomography at the time of rebleeding showed an increase in the hematoma and extravasation in the peripheral right lateral thoracic artery. The patient was immediately treated with emergency angiography, and coil embolization was performed. After this treatment, the patient has done well and there has been no subsequent recurrence of bleeding.

EP4-induced mitochondrial localization and cell migration mediated by CALML6 in human oral squamous cell carcinoma.

Lymph node metastasis, primarily caused by the migration of oral squamous cell carcinoma (OSCC) cells, stands as a crucial prognostic marker. We have previously demonstrated that EP4, a subtype of the prostaglandin E2 (PGE2) receptor, orchestrates OSCC cell migration via Ca2+ signaling. The exact mechanisms by which EP4 influences cell migration through Ca2+ signaling, however, is unclear. Our study aims to clarify how EP4 controls OSCC cell migration through this pathway. We find that activating EP4 with an agonist (ONO-AE1-473) increased intracellular Ca2+ levels and the migration of human oral cancer cells (HSC-3), but not human gingival fibroblasts (HGnF). Further RNA sequencing linked EP4 to calmodulin-like protein 6 (CALML6), whose role remains undefined in OSCC. Through protein-protein interaction network analysis, a strong connection is identified between CALML6 and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), with EP4 activation also boosting mitochondrial function. Overexpressing EP4 in HSC-3 cells increases experimental lung metastasis in mice, whereas inhibiting CaMKK2 with STO-609 markedly lowers these metastases. This positions CaMKK2 as a potential new target for treating OSCC metastasis. Our findings highlight CALML6 as a pivotal regulator in EP4-driven mitochondrial respiration, affecting cell migration and metastasis via the CaMKK2 pathway.

A Case of Myxoid Pleomorphic Liposarcoma with Rhabdoid Cells: A Diagnostic Pitfall.

Myxoid pleomorphic liposarcoma (MPLS) is an extremely rare tumor listed in the fifth edition of the WHO classification (2020). Histologically, it mainly comprises a mixture of myxoid and pleomorphic liposarcoma-like components. Genetically, it lacks FUS/EWSR1::DDIT3 fusion and MDM2 amplification. Herein, we describe an example of MPLS with rhabdoid cells in a 10-year-old girl who presented with a growing mass in the right inguinal region. The specimen from the wide excision measured 68 mm × 55 mm × 43 mm, and a circumscribed and lobulated mass was observed in the subcutaneous tissue. Histologically, oval-to-short, spindle-shaped, proliferating tumor cells with moderate nuclear atypia and mesh-like capillaries against a myxoid background were noted. Adipocytes were observed focally, while rhabdoid cells were observed multifocally. Immunohistochemically, the tumor showed inconsistent reactivity for desmin but was negative for MYOD1, myogenin, MDM2, and CDK4. Fluorescence in situ hybridization revealed no DDIT3 rearrangement. Despite adjuvant chemotherapy, the tumor metastasized to the thoracic cavity 24 months after excision. The metastatic lesions contained abundant lipoblasts rather than rhabdoid cells, and we concluded this tumor was a MPLS. The presence of rhabdoid cells could be a diagnostic pitfall, and recognizing such a variation in histology would help improve diagnostic accuracy.

Favourable surgical outcomes for either second primary lung cancer or intrapulmonary metastasis after resection of non-small-cell lung cancer.

OBJECTIVES: Metachronous lung cancer arising after resection of non-small-cell lung cancer is either a second primary lung cancer (SPLC) or intrapulmonary metastasis (IPM) of the initial lung cancer; however, differential diagnosis is difficult. We evaluated the surgical outcomes of metachronous lung cancer in a combined population of patients with SPLC and IPM. METHODS: A retrospective study of 3534 consecutive patients with resected non-small-cell lung cancer between 1992 and 2016 was conducted at 4 institutions. RESULTS: A total of 105 patients (66 males; median age, 70 years) who underwent a second pulmonary resection for metachronous lung cancer were included. Most patients (81%) underwent sublobar resection, and there was no 30-day mortality. All metachronous lung cancers were cN0, 5 were pN1-2. The postoperative comprehensive histologic assessment revealed SPLC (n = 77) and IPM (n = 28). The 5-year overall survival rate after the second resection was 70.6% (median follow-up: 69.7 months). A multivariable analysis showed that age >70 years at the second resection (P = 0.013), male sex (P = 0.003), lymph node involvement in metachronous cancer (P < 0.001), pathological invasive size of metachronous cancer >15 mm (P < 0.001) and overlapping squamous cell carcinoma histology of the initial and metachronous cancers (P = 0.003) were significant prognostic factors for poor survival after the second resection, whereas histological IPM was not (P = 0.065). CONCLUSIONS: Surgery for cN0 metachronous lung cancer is safe and shows good outcomes. There were no statistically significant differences in the SPLC and IPM results. Caution should be exercised when operating on patients with overlapping squamous cell carcinoma.

Hydrostatic pressure under hypoxia facilitates fabrication of tissue-engineered vascular grafts derived from human vascular smooth muscle cells in vitro.

Biologically compatible vascular grafts are urgently required. The scaffoldless multi-layered vascular wall is considered to offer theoretical advantages, such as facilitating cells to form cell-cell and cell-matrix junctions and natural extracellular matrix networks. Simple methods are desired for fabricating physiological scaffoldless tissue-engineered vascular grafts. Here, we showed that periodic hydrostatic pressurization under hypoxia (HP/HYP) facilitated the fabrication of multi-layered tunica media entirely from human vascular smooth muscle cells. Compared with normoxic atmospheric pressure, HP/HYP increased expression of N-myc downstream-regulated 1 (NDRG1) and the collagen-cross-linking enzyme lysyl oxidase in human umbilical artery smooth muscle cells. HP/HYP increased N-cadherin-mediated cell-cell adhesion via NDRG1, cell-matrix interaction (i.e., clustering of integrin α5ß1 and fibronectin), and collagen fibrils. We then fabricated vascular grafts using HP/HYP during repeated cell seeding and obtained 10-layered smooth muscle grafts with tensile rupture strength of 0.218-0.396 MPa within 5 weeks. Implanted grafts into the rat aorta were endothelialized after 1 week and patent after 5 months, at which time most implanted cells had been replaced by recipient-derived cells. These results suggest that HP/HYP enables fabrication of scaffoldless human vascular mimetics that have a spatial arrangement of cells and matrices, providing potential clinical applications for cardiovascular diseases. STATEMENT OF SIGNIFICANCE: Tissue-engineered vascular grafts (TEVGs) are theoretically more biocompatible than prosthetic materials in terms of mechanical properties and recipient cell-mediated tissue reconstruction. Although some promising results have been shown, TEVG fabrication processes are complex, and the ideal method is still desired. We focused on the environment in which the vessels develop in utero and found that mechanical loading combined with hypoxia facilitated formation of cell-cell and cell-matrix junctions and natural extracellular matrix networks in vitro, which resulted in the fabrication of multi-layered tunica media entirely from human umbilical artery smooth muscle cells. These scaffoldless TEVGs, produced using a simple process, were implantable and have potential clinical applications for cardiovascular diseases.

Local Net Charge State of Collagen Triple Helix Is a Determinant of FKBP22 Binding to Collagen III.

Mutations in the FKBP14 gene encoding the endoplasmic reticulum resident collagen-related proline isomerase FK506 binding protein 22 kDa (FKBP22) result in kyphoscoliotic Ehlers-Danlos Syndrome (EDS), which is characterized by a broad phenotypic outcome. A plausible explanation for this outcome is that FKBP22 participates in the biosynthesis of subsets of collagen types: FKBP22 selectively binds to collagens III, IV, VI, and X, but not to collagens I, II, V, and XI. However, these binding mechanisms have never been explored, and they may underpin EDS subtype heterogeneity. Here, we used collagen Toolkit peptide libraries to investigate binding specificity. We observed that FKBP22 binding was distributed along the collagen helix. Further, it (1) was higher on collagen III than collagen II peptides and it (2) was correlated with a positive peptide charge. These findings begin to elucidate the mechanism by which FKBP22 interacts with collagen.

Physiological functions of calcium signaling via Orai1 in cancer.

Intracellular calcium (Ca2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca2+ entry (SOCE) is a major mechanism by which Ca2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca2+ entry (SOCE) is also a receptor-regulated Ca2+ entry pathway that maintains Ca2+ homeostasis by sensing reduced Ca2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors located in the ER membrane. The, IP3 receptors in the ER membrane trigger a rapid and transient release of Ca2+ from the ER store. The resulting depletion of ER Ca2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca2+ channel subunits (also known as CRACM1) on the PM, leading to Ca2+ influx from the extracellular space to increase intracellular Ca2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca2+ release-activated Ca2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial-mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings.

Regional differences in epidermal growth factor receptor-tyrosine kinase inhibitor therapy in lung cancer treatment using a national database in Japan.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are extensively used in the treatment of non-small cell lung cancer (NSCLC); hence, equal access to them is important. Therefore, this study aimed to identify regional differences in the prescription of EGFR-TKIs and the factors contributing to these differences. In this ecological study, we collected data using the National Database Open Data and the National Cancer Registry. The standardized claim ratio (SCR) was used as an indicator of the number of EGFR-TKI prescriptions. Additionally, we examined the association between SCR and various factors to identify the factors associated with this difference. The average SCR for the top three provinces was 153.4, while the average for the bottom three provinces was 61.6. Multivariate analysis used for evaluating the association of SCR with variables revealed that the number of designated cancer hospitals and radiation therapies were independent factors associated with the SCR of EGFR-TKIs. There were significant regional differences in the prescriptions of EGFR-TKIs in Japan based on the number of coordinated designated cancer hospitals and the number of patients receiving radiotherapy alone. These findings emphasize the need to implement policies to increase the number of hospitals to reduce regional differences.

Relationship between Cognitive Function and Sway of Body in Standing Posture: A Cross-Sectional Study.

BACKGROUND: The influence of neurological or balance dysfunction on cognitive impairment has not been well studied. We compared the results of the balance test, measured by either head or foot sway to consider whole body sway, with those of the cognitive impairment test. METHODS: Individuals of either gender, aged over 60 years, underwent a 30 s balance test. We measured sway while standing on one-leg or two-legs. Sway was evaluated by the distance or area of movement of the head or foot pressure. We also evaluated the effect of visual condition: eyes-open (EO) or -closed (EC). The Mini-Mental State Examination (MMSE) was used to evaluate the degree of cognitive impairment. RESULTS: The head sway area standing on one leg was significantly correlated to MMSE score with EO (correlation r = -0.462). In standing on two legs, no sway test results showed a significant correlation to MMSE scores with EO. With EC, the magnitude of sway became greater, and was significantly correlated to MMSE scores in the head distance. CONCLUSION: Although the correlation between head sway and MMSE was not strong, head sway showed a stronger correlation than did foot pressure sway. Standing on one leg, as measured by head sway area, may thus predict cognitive impairment.

Store-operated calcium entry via ORAI1 regulates doxorubicin-induced apoptosis and prevents cardiotoxicity in cardiac fibroblasts.

Despite exhibiting cardiotoxicity, doxorubicin (DOX) is widely used for cancer treatments. Cardiac fibroblasts (CFs) are important in the pathogenesis of heart failure. This necessitates the study of the effect of DOX on CFs. The impairment of calcium (Ca2+) homeostasis is a common mechanism of heart failure. Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2⁺ entry pathway that maintains calcium balance by sensing reduced calcium stores in the endoplasmic reticulum. ORAI1, a calcium channel protein and the most important component of SOCE, is highly expressed in human cardiac fibroblasts (HCFs). It is upregulated in CFs from failing ventricles. However, whether ORAI1 in HCFs is increased and/or plays a role in DOX-induced cardiotoxicity remains unknown. In this study, we aimed to elucidate the relationship between ORAI1/SOCE and DOX-induced heart failure. Induction of apoptosis by DOX was characterized in HCFs. Apoptosis and cell cycle analyses were performed by fluorescence-activated cell sorting (FACS). Reactive oxygen species (ROS) production was measured using fluorescence. YM-58483 was used as an ORAI1/SOCE inhibitor. ORAI1-knockdown cells were established by RNA interference. In vivo experiments were performed by intraperitoneally injecting YM-58483 and DOX into mice. We first demonstrated that DOX significantly increased the protein expression level of p53 in HCFs by western blotting. FACS analysis revealed that DOX increased early apoptosis and induced cell cycle arrest in the G2 phase in fibroblasts. DOX also increased ROS production. DOX significantly increased the expression level of ORAI1 in CFs. Both YM-58483 and ORAI1 gene knockdown attenuated DOX-induced apoptosis. Similarly, YM-58483 attenuated cell cycle arrest in the G2 phase, and ORAI1 knockdown attenuated DOX-induced ROS production in HCFs. In the animal experiment, YM-58483 attenuated DOX-induced apoptosis. In HCFs, ORAI1/SOCE regulates p53 expression and plays an important role in DOX-induced cardiotoxicity. ORAI1 may serve as a new target for preventing DOX-induced heart failure.

Lysyl hydroxylase 3-mediated post-translational modifications are required for proper biosynthesis of collagen α1α1α2(IV).

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome.

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis.

Antibodies are central effectors of the adaptive immune response, widespread used therapeutics, but also potentially disease-causing biomolecules. Antibody folding catalysts in the plasma cell are incompletely defined. Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease with increasingly recognized autoimmune features. We found elevated expression of FK506-binding protein 11 (FKBP11) in IPF lungs where FKBP11 specifically localized to antibody-producing plasma cells. Suggesting a general role in plasma cells, plasma cell-specific FKBP11 expression was equally observed in lymphatic tissues, and in vitro B cell to plasma cell differentiation was accompanied by induction of FKBP11 expression. Recombinant human FKBP11 was able to refold IgG antibody in vitro and inhibited by FK506, strongly supporting a function as antibody peptidyl-prolyl cis-trans isomerase. Induction of ER stress in cell lines demonstrated induction of FKBP11 in the context of the unfolded protein response in an X-box-binding protein 1 (XBP1)-dependent manner. While deficiency of FKBP11 increased susceptibility to ER stress-mediated cell death in an alveolar epithelial cell line, FKBP11 knockdown in an antibody-producing hybridoma cell line neither induced cell death nor decreased expression or secretion of IgG antibody. Similarly, antibody secretion by the same hybridoma cell line was not affected by knockdown of the established antibody peptidyl-prolyl isomerase cyclophilin B. The results are consistent with FKBP11 as a novel XBP1-regulated antibody peptidyl-prolyl cis-trans isomerase and indicate significant redundancy in the ER-resident folding machinery of antibody-producing hybridoma cells.

Sequence-dependent mechanics of collagen reflect its structural and functional organization.

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagen proteins. In particular, network-forming collagen IV, an integral component of basement membranes, has been far less studied than fibril-forming collagens. A key feature of collagen IV is the presence of interruptions in the triple-helix-defining (Gly-X-Y) sequence along its collagenous domain. Here, we used atomic force microscopy to determine the impact of sequence heterogeneity on the local flexibility of collagen IV and of the fibril-forming collagen III. Our extracted flexibility profile of collagen IV reveals that it possesses highly heterogeneous mechanics, ranging from semiflexible regions as found for fibril-forming collagens to a lengthy region of high flexibility toward its N-terminus. A simple model in which flexibility is dictated only by the presence of interruptions fit the extracted profile reasonably well, providing insight into the alignment of chains and demonstrating that interruptions, particularly when coinciding in multiple chains, significantly enhance local flexibility. To a lesser extent, sequence variations within the triple helix lead to variable flexibility, as seen along the continuously triple-helical collagen III. We found this fibril-forming collagen to possess a high-flexibility region around its matrix-metalloprotease binding site, suggesting a unique mechanical fingerprint of this region that is key for matrix remodeling. Surprisingly, proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are unlikely to control its compaction during secretion. Although extracellular chloride ions play a role in triggering collagen IV network formation, they do not appear to modulate the structure of its collagenous domain.

Increased Plasma Levels of Myosin Heavy Chain 11 Is Associated with Atherosclerosis.

Many studies have revealed numerous potential biomarkers for atherosclerosis, but tissue-specific biomarkers are still needed. Recent lineage-tracing studies revealed that smooth muscle cells (SMCs) contribute substantially to plaque formation, and the loss of SMCs causes plaque vulnerability. We investigated the association of SMC-specific myosin heavy chain 11 (myosin-11) with atherosclerosis. Forty-five patients with atherosclerosis and 34 control subjects were recruited into our study. In the atherosclerosis patients, 35 patients had either coronary artery disease (CAD) or peripheral artery disease (PAD), and 10 had both CAD and PAD. Coronary arteries isolated from five patients were subjected to histological study. Circulating myosin-11 levels were higher in the CAD or PAD group than in controls. The area under the receiver operating characteristic curve of myosin-11 was 0.954. Circulating myosin-11 levels in the CAD and PAD group were higher than in the CAD or PAD group, while high-sensitivity C-reactive protein concentrations did not differ between these groups. Multinomial logistic regression analyses showed a significant association of myosin-11 levels with the presence of multiple atherosclerotic regions. Myosin-11 was expressed in the medial layer of human atherosclerotic lesions where apoptosis elevated. Circulating myosin-11 levels may be useful for detecting spatial expansion of atherosclerotic regions.

[Doxorubicin directly induced fibrotic change of cardiac fibroblasts].

Doxorubicin (DOX)-induced cardiomyopathy has a poor prognosis. No early detection or effective treatment methods are available in clinical. The mechanisms of cardiotoxicity were considered as oxidative stress and apoptosis in cardiomyocytes. However, the effect of DOX on cardiac fibroblasts remains to be developed. We investigated the direct effect of DOX on the function of human cardiac fibroblasts (HCFs) independently of cell death pathway. Animal study showed that lower dose of DOX (4 mg/kg/week for 3 weeks, i.p.) than a toxic cumulate dose, induced perivascular fibrosis without cell death in hear of mice. DOX increased the protein expression of α-SMA (a marker of trans-differentiation) in HCFs culture cells, indicating that DOX promoted the trans-differentiation of HCFs into myofibroblast. DOX also increased the mRNA and protein expression of matrix metalloproteinase (MMP)-1 in less than 0.1 µM which did not induce cell apoptosis of HCFs cells via PI3K/Akt pathway in HCFs. DOX increased Interleukin-6 (IL-6) via transforming growth factor (TGF)-ß/Smad pathway. In addition, DOX induced the mitochondrial damage and increased the expression of Interleukin-1 (IL-1) via stress-activated protein kinases (SAPK)/ c-Jun NH-2termial kinase (JNK). A peroxisome proliferator-activated receptor gamma (PPARγ) agonist, pioglitazone hydrochloride attenuated the expression of fibrotic marker such as α-SMA and galectin-3 and collagen1 via SAPK/JNK signaling. Pioglitazone also suppressed DOX-induced early fibrotic response in vivo. In conclusion, these findings suggested that low dose DOX induced reactive fibrotic change of cardiac fibroblasts via cell death-independent pathway. There may be potentially new mechanisms of DOX induced cardiotoxicity in clinical usage.

[Postoperative Relapse of Combined Large‒Cell Neuroendocrine Carcinoma of the Lung with a Remarkable Transient Response to Amrubicin Monotherapy in an Elderly Patient-A Case Report].

An 86‒year‒old man with chronic kidney disease underwent surgical resection for combined large‒cell neuroendocrine carcinoma of the left lower lobe of the lung(pT2aN1M0, stage ⅡB). Five months later, multiple liver and bone metastases and mediastinal lymph node recurrence were detected. After 9 courses of amrubicin monotherapy(32 mg/m2 for 3 consecutive days), his tumor marker levels normalized, and radiological examination revealed a complete tumor response. Adverse events occurred, but they were tolerable except a decrease in the neutrophil count. The patient remained in good condition for several months but died of tumor relapse 22 months after the initial recurrence. Amrubicin monotherapy was considered to be one of the treatment choices for recurrent large‒cell neuroendocrine carcinoma of the lung in elderly patients.

Multilayered Human Skeletal Muscle Myoblast Sheets Promote the Healing Process After Colonic Anastomosis in Rats.

Colorectal anastomotic leakage is one of the most feared and fatal complications of colorectal surgery. To date, no external coating material that can prevent anastomotic leakage has been developed. As myoblasts possess anti-inflammatory capacity and improve wound healing, we developed a multilayered human skeletal muscle myoblast (HSMM) sheet by periodic exposure to supraphysiological hydrostatic pressure during repeated cell seeding. We assessed whether the application of an HSMM sheet can promote the healing process after colonic anastomosis. Partial colectomy and insufficient suturing were employed to create a high-risk colo-colonic anastomosis model in 60 nude rats. Rats were divided into a control group (n = 30) and an HSMM sheet group (n = 30). Macroscopic findings, anastomotic bursting pressure, and histology at the colonic anastomotic site were evaluated on postoperative day (POD) 3, 5, 7, 14, and 28. The application of an HSMM sheet significantly suppressed abscess formation at the anastomotic site compared to the control group on POD3 and 5. The anastomotic bursting pressure in the HSMM sheet group was higher than that in the control group on POD3 and 5. Inflammatory cell infiltration in the HSMM sheet group was significantly suppressed compared to that in the control group throughout the time course. Collagen deposition in the HSMM sheet group on POD3 was significantly abundant compared to that in the control group. Regeneration of the mucosa at the colonic anastomotic site was promoted in the HSMM sheet group compared to that in the control group on POD14 and 28. Immunohistochemical analysis demonstrated that surviving cells in the HSMM sheet gradually decreased with postoperative time and none were detected on POD14. These results suggest that the application of a multilayered HSMM sheet may prevent postoperative colonic anastomotic leakage.

Aberrant binding of mutant HSP47 affects posttranslational modification of type I collagen and leads to osteogenesis imperfecta.

Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.