The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics.

The prevalence of head and neck squamous cell carcinoma (HNSCC) has continued to rise for decades. However, drug resistance to chemotherapeutics and relapse, mediated by cancer stem cells (CSCs), remains a significant impediment in clinical oncology to achieve successful treatment. Therefore, we focused on analyzing CSCs in HNSCC and demonstrated the effect of melatonin (Mel) and verteporfin (VP) on SCC-25 cells. HNSCC CSCs were enriched in the reactive oxygen species-low state and in sphere-forming cultures. Combination treatment with Mel and VP decreased HNSCC viability and increased apoptosis without causing significant damage to normal cells. Sphere-forming ability and stem cell population were reduced by co-treatment with Mel and VP, while mitochondrial ROS level was increased by the treatment. Furthermore, the expression of mitophagy markers, parkin and PINK1, was significantly decreased in the co-treated cells. Mel and VP induced mitochondrial depolarization and inhibited mitochondrial function. Parkin/TOM20 was localized near the nucleus and formed clusters of mitochondria in the cells after treatment. Moreover, Mel and VP downregulated the expression of markers involved in epithelial-mesenchymal transition and metastasis. The migration capacity of cells was significantly decreased by co-treatment with Mel and VP, accompanied by the down-regulation of MMP-2 and MMP-9 expression. Taken together, these results indicate that co-treatment with Mel and VP induces mitochondrial dysfunction, resulting in the apoptosis of CSCs. Mel and VP could thus be further investigated as potential therapies for HNSCC through their action on CSCs.

Implication of Echinochrome A in the Plasticity and Damage of Intestinal Epithelium.

The diverse therapeutic feasibility of the sea urchin-derived naphthoquinone pigment, Echinochrome A (Ech A), has been studied. Simple and noninvasive administration routes should be explored, to obtain the feasibility. Although the therapeutic potential has been proven through several preclinical studies, the biosafety of orally administered Ech A and its direct influence on intestinal cells have not been evaluated. To estimate the bioavailability of Ech A as an oral administration drug, small intestinal and colonic epithelial organoids were developed from mice and humans. The morphology and cellular composition of intestinal organoids were evaluated after Ech A treatment. Ech A treatment significantly increased the expression of LGR5 (~2.38-fold change, p = 0.009) and MUC2 (~1.85-fold change, p = 0.08). Notably, in the presence of oxidative stress, Ech A attenuated oxidative stress up to 1.8-fold (p = 0.04), with a restored gene expression of LGR5 (~4.11-fold change, p = 0.0004), as well as an increased expression of Ly6a (~3.51-fold change, p = 0.005) and CLU (~2.5-fold change, p = 0.01), markers of revival stem cells. In conclusion, Ech A is harmless to intestinal tissues; rather, it promotes the maintenance and regeneration of the intestinal epithelium, suggesting possible beneficial effects on the intestine when used as an oral medication.

Metformin Protects the Intestinal Barrier by Activating Goblet Cell Maturation and Epithelial Proliferation in Radiation-Induced Enteropathy.

Radiotherapy or accidental exposure to high-dose radiation can cause severe damage to healthy organs. The gastrointestinal (GI) tract is a radiation-sensitive organ of the body. The intestinal barrier is the first line of defense in the GI tract, and consists of mucus secreted by goblet cells and a monolayer of epithelium. Intestinal stem cells (ISCs) help in barrier maintenance and intestinal function after injury by regulating efficient regeneration of the epithelium. The Wnt/ß-catenin pathway plays a critical role in maintaining the intestinal epithelium and regulates ISC self-renewal. Metformin is the most widely used antidiabetic drug in clinical practice, and its anti-inflammatory, antioxidative, and antiapoptotic effects have also been widely studied. In this study, we investigated whether metformin alleviated radiation-induced enteropathy by focusing on its role in protecting the epithelial barrier. We found that metformin alleviated radiation-induced enteropathy, with increased villi length and crypt numbers, and restored the intestinal barrier function in the irradiated intestine. In a radiation-induced enteropathy mouse model, metformin treatment increased tight-junction expression in the epithelium and inhibited bacterial translocation to mesenteric lymph nodes. Metformin increased the number of ISCs from radiation toxicity and enhanced epithelial repair by activating Wnt/ß-catenin signaling. These data suggested that metformin may be a potential therapeutic agent for radiation-induced enteropathy.

Combined Administration of Pravastatin and Metformin Attenuates Acute Radiation-Induced Intestinal Injury in Mouse and Minipig Models.

Radiation-induced gastrointestinal (GI) damage is one of the critical factors that serve as basis for the lethality of nuclear accidents or terrorism. Further, there are no Food and Drug Administration-approved agents available to mitigate radiation-induced intestinal injury. Although pravastatin (PS) has been shown to exhibit anti-inflammatory and epithelial reconstructive effects following radiation exposure using mouse and minipig models, the treatment failed to improve the survival rate of high-dose irradiated intestinal injury. Moreover, we previously found that metformin (MF), a common drug used for treating type 2 diabetes mellitus, has a mitigating effect on radiation-induced enteropathy by promoting stem cell properties. In this study, we investigated whether the combined administration of PS and MF could achieve therapeutic effects on acute radiation-induced intestinal injury in mouse and minipig models. We found that the combined treatment markedly increased the survival rate and attenuated histological damage in a radiation-induced intestinal injury mouse model, in addition to epithelial barrier recovery, anti-inflammatory effects, and improved epithelial proliferation with stem cell properties. Furthermore, in minipig models, combined treatment with PS and MF ameliorates gross pathological damage in abdominal organs and attenuated radiation-induced intestinal histological damage. Therefore, the combination of PS and MF effectively alleviated radiation-induced intestinal injury in the mouse and minipig models. We believe that the combined use of PS and MF is a promising therapeutic approach for treating radiation-induced intestinal injury.

16-channel tunable and 25-Gb/s EAM-integrated DBR-LD for WDM-based mobile front-haul networks.

We report a tunable distributed Bragg reflector-laser diode (DBR-LD) integrated with an electro-absorption-modulator (EAM) at an operating wavelength of 1.3 µm. This LD consists of gain, phase control (PC), DBR, and EAM sections, realized by using a butt-coupling technique in monolithically integrating the multiple quantum wells (MQWs) with the passive core and by applying an etched-mesa buried hetero-structure (EMBH) to the resonance cavity (i.e., gain to DBR section) and a deep-ridge type to the EAM section in fabricating the waveguide structure. Wavelength tuning of the LD is achieved by both applying a voltage to the heater metal of DBR section (coarse tuning) and injecting a current to the ohmic metal of PC section (fine tuning). From the work, the fabricated chips show a threshold current of about 13 mA, a side mode suppression ratio (SMSR) of more than 35 dB, and a tuning range of 15 nm within a heater voltage of 2 V. Dynamic tests for the EAM-integrated LD show the 3 dB bandwidth of more than 20 GHz and clear 25 Gb/s eye openings with a dynamic extinction ratio (DER) of over 7 dB for 16 channels spaced at the wavelength interval of 0.55 nm. Based on these results, we conclude that the EAM-integrated DBR-LD is capable of providing 16 channel operation at a data rate of 25 Gb/s and can be used as an effective light source for WDM-based mobile front-haul networks.

Association Between Gout and Dementia in the Elderly: A Nationwide Population-Based Cohort Study.

OBJECTIVE: The data showing the association between gout and dementia are inconsistent. The objective of this study was to examine whether gout is associated with the risk of dementia in the elderly. METHODS: This retrospective cohort study used population-based representative claims data from the National Health Insurance Service in Korea. We used the Elderly Cohort database which represents 10% of the elderly Koreans over the age of 60, from 2002 to 2013. We assessed the association of gout with a new diagnosis of dementia with Cox proportional hazard models and adjusted the data for potential covariates such as demographics (age, sex) and comorbidities. RESULTS: We included 22,178 patients with gout and 113,590 without. In each group, 2,557 (11.53%) and 18,264 (16.08%) patients, respectively, had dementia. In multivariable analyses, gout was independently associated with a significantly lower hazard ratio of incident dementia, with an adjusted hazard ratio of 0.63 (95% CI, 0.60-0.66). A sub-group analysis conducted to find out the effects of gout medication showed that febuxostat use significantly decreased incident dementia. CONCLUSION: Gout was independently associated with a 37% lower risk of dementia in the elderly.

Pediatric Cannabis Single-Substance Exposures Reported to the Michigan Poison Center From 2008-2019 After Medical Marijuana Legalization.

BACKGROUND: Legalization of medical and recreational cannabis is a major contributor to pediatric cannabis exposures. The trends and magnitude of pediatric cannabis exposures in Michigan after medical cannabis legalization in 2008 have not been assessed. OBJECTIVE: To describe the temporal trends of pediatric cannabis exposures reported to the Michigan Poison Center (MiPC) after medical cannabis was legalized in 2008 and 1 year after legalization of recreational cannabis in 2018. METHODS: Retrospective electronic chart review of pediatric (<18 years old) single-substance cannabis exposures reported to the MiPC from January 1, 2008 to December 31, 2019. Routes of cannabis exposure were reported as ingestion, inhalation, and unknown. Types of ingested cannabis products were also documented. RESULTS: Between 2008 and 2019, 426 pediatric cannabis single exposures were reported. The median patient age was 6.0 years (interquartile range 2-15 years). Age distribution was bimodal. A total of 327 (76.8%) exposures were from cannabis ingestion, 79 (18.5%) from inhalation, 2 (0.5%) from both ingestion and inhalation, and 18 (4.2%) from unknown route. The doubling time for number of cases was 2.1 years, and the total number of annual reported cases increased after 2016. Teenagers (13-17 years) had the highest number of inhalational exposures, whereas young children (0-5 years) had the highest number of ingestions. CONCLUSION: Single-substance pediatric cannabis exposures reported to the Michigan Poison Center increased after medical cannabis was legalized in 2008 through recreational legalization in 2018.

Long-Lasting Growth Hormone Regulated by the Ubiquitin-Proteasome System.

To increase the half-life of growth hormones, we proposed its long-lasting regulation through the ubiquitin-proteasome system (UPS). We identified lysine residues (K67, K141, and K166) that are involved in the ubiquitination of human growth hormone (hGH) using ubiquitination site prediction programs to validate the ubiquitination sites, and then substituted these lysine residues with arginine residues. We identified the most effective substituent (K141R) to prevent ubiquitination and named it AUT-hGH. hGH was expressed and purified in the form of hGH-His, and ubiquitination was first verified at sites containing K141 in the blood stream. Through the study, we propose that AUT-hGH with an increased half-life could be used as a long-lasting hGH in the blood stream.

Aesculetin Accelerates Osteoblast Differentiation and Matrix-Vesicle-Mediated Mineralization.

The imbalance between bone resorption and bone formation in favor of resorption results in bone loss and deterioration of bone architecture. Osteoblast differentiation is a sequential event accompanying biogenesis of matrix vesicles and mineralization of collagen matrix with hydroxyapatite crystals. Considerable efforts have been made in developing naturally-occurring plant compounds, preventing bone pathologies, or enhancing bone regeneration. Coumarin aesculetin inhibits osteoporosis through hampering the ruffled border formation of mature osteoclasts. However, little is known regarding the effects of aesculetin on the impairment of matrix vesicle biogenesis. MC3T3-E1 cells were cultured in differentiation media with 1-10 µM aesculetin for up to 21 days. Aesculetin boosted the bone morphogenetic protein-2 expression, and alkaline phosphatase activation of differentiating MC3T3-E1 cells. The presence of aesculetin strengthened the expression of collagen type 1 and osteoprotegerin and transcription of Runt-related transcription factor 2 in differentiating osteoblasts for 9 days. When ≥1-5 µM aesculetin was added to differentiating cells for 15-18 days, the induction of non-collagenous proteins of bone sialoprotein II, osteopontin, osteocalcin, and osteonectin was markedly enhanced, facilitating the formation of hydroxyapatite crystals and mineralized collagen matrix. The induction of annexin V and PHOSPHO 1 was further augmented in ≥5 µM aesculetin-treated differentiating osteoblasts for 21 days. In addition, the levels of tissue-nonspecific alkaline phosphatase and collagen type 1 were further enhanced within the extracellular space and on matrix vesicles of mature osteoblasts treated with aesculetin, indicating matrix vesicle-mediated bone mineralization. Finally, aesculetin markedly accelerated the production of thrombospondin-1 and tenascin C in mature osteoblasts, leading to their adhesion to preformed collagen matrix. Therefore, aesculetin enhanced osteoblast differentiation, and matrix vesicle biogenesis and mineralization. These findings suggest that aesculetin may be a potential osteo-inductive agent preventing bone pathologies or enhancing bone regeneration.

Dietary Collagen Hydrolysates Ameliorate Furrowed and Parched Skin Caused by Photoaging in Hairless Mice.

Collagen hydrolysates have been suggested as a favorable antiaging modality in skin photoaged by persistent exposure to ultraviolet radiation (UV). The current study evaluated the beneficial effect of collagen hydrolysates (fsCH) extracted from Pangasius hypophthalmus fish skin on wrinkle formation and moisture preservation in dorsal skin of hairless mice challenged with UV-B. Inter-comparative experiments were conducted for anti-photoaging among fsCH, retinoic acid (RA), N-acetyl-D-glucosamine (NAG), and glycine-proline-hydroxyproline (GPH). Treating human HaCaT keratinocytes with 100-200 µg/mL fsCH reciprocally ameliorated the expression of aquaporin 3 (AQP3) and CD44 deranged by UV-B. The UV-B-induced deep furrows and skin thickening were improved in parched dorsal skin of mice supplemented with 206-412 mg/kg fsCH as well as RA and GPH. The UV-B irradiation enhanced collagen fiber loss in the dorsal dermis, which was attenuated by fsCH through enhancing procollagen conversion to collagen. The matrix metalloproteinase expression by UV-B in dorsal skin was diminished by fsCH, similar to RA and GPH, via blockade of collagen degradation. Supplementing fsCH to UV-B-irradiated mice decreased transepidermal water loss in dorsal skin with reduced AQP3 level and restored keratinocyte expression of filaggrin. The expression of hyaluronic acid synthase 2 and hyaluronidase 1 by UV-B was remarkably ameliorated with increased production of hyaluronic acid by treating fsCH to photoaged mice. Taken together, fsCH attenuated photoaging typical of deep wrinkles, epidermal thickening, and skin water loss, like NAG, RA, or GPH, through inhibiting collagen destruction and epidermal barrier impairment.

Astragalin Inhibits Cigarette Smoke-Induced Pulmonary Thrombosis and Alveolar Inflammation and Disrupts PAR Activation and Oxidative Stress-Responsive MAPK-Signaling.

Epidemiological evidence shows that smoking causes a thrombophilic milieu that may play a role in the pathophysiology of chronic obstructive pulmonary disease (COPD) as well as pulmonary thromboembolism. The increased nicotine level induces a prothrombotic status and abnormal blood coagulation in smokers. Since several anticoagulants increase bleeding risk, alternative therapies need to be identified to protect against thrombosis without affecting hemostasis. Astragalin is a flavonoid present in persimmon leaves and green tea seeds and exhibits diverse activities of antioxidant and anti-inflammation. The current study investigated that astragalin attenuated smoking-induced pulmonary thrombosis and alveolar inflammation. In addition, it was explored that molecular links between thrombosis and inflammation entailed protease-activated receptor (PAR) activation and oxidative stress-responsive mitogen-activated protein kinase (MAPK)-signaling. BALB/c mice were orally administrated with 10-20 mg/kg astragalin and exposed to cigarette smoke for 8 weeks. For the in vitro study, 10 U/mL thrombin was added to alveolar epithelial A549 cells in the presence of 1-20 µM astragalin. The cigarette smoking-induced the expression of PAR-1 and PAR-2 in lung tissues, which was attenuated by the administration of ≥10 mg/kg astragalin. The oral supplementation of ≥10 mg/kg astragalin to cigarette smoke-challenged mice attenuated the protein induction of urokinase plasminogen activator, plasminogen activator inhibitor-1and tissue factor, and instead enhanced the induction of tissue plasminogen activator in lung tissues. The astragalin treatment alleviated cigarette smoke-induced lung emphysema and pulmonary thrombosis. Astragalin caused lymphocytosis and neutrophilia in bronchoalveolar lavage fluid due to cigarette smoke but curtailed infiltration of neutrophils and macrophages in airways. Furthermore, this compound retarded thrombin-induced activation of PAR proteins and expression of inflammatory mediators in alveolar cells. Treating astragalin interrupted PAR proteins-activated reactive oxygen species production and MAPK signaling leading to alveolar inflammation. Accordingly, astragalin may interrupt the smoking-induced oxidative stress-MAPK signaling-inflammation axis via disconnection between alveolar PAR activation and pulmonary thromboembolism.

Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft.

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Mid-Aortic Syndrome in Williams-Beuren Syndrome with an Atypical Small-Sized Deletion of Chromosome 7q11.23 Misdiagnosed as Takayasu Arteritis.

Mid-aortic syndrome (MAS) is a rare condition characterized by stenosis of the distal thoracic and/or abdominal aorta. Williams-Beuren syndrome (WBS) is a relatively rare cause of MAS. We report a case of incidentally diagnosed MAS caused by WBS without typical manifestations caused by an atypical small-sized deletion in chromosome 7q11.23, which was initially misdiagnosed as Takayasu arteritis.

Development of molecular markers to distinguish between morphologically similar edible plants and poisonous plants using a real-time PCR assay.

BACKGROUND: As a result of similar appearances between edible and poisonous plants, 42 patients have ingested poisonous plants from 2013 to 2017 in Korea. We have developed species-specific primer sets of three of edible and poisonous plants sets (Ligularia fischeri & Caltha palustris, Artemisia annua & Ambrosia artemisiifolia and Hemerocallis fulva & Veratrum maackii) for distinguishing both plants using a real-time polymerase chain reaction assay. RESULTS: The efficiencies of the developed primer sets ranged from 87.8% to 102.0%. The developed primer sets have significant correlation coefficient values between the Ct values and the log DNA concentration for their target species (r2 > 0.99). The cut-off lines as the crossing point values of the limit of quantitation of the target species were determined, and all non-target species were amplified later than the cut-off cycles. Then, the effectiveness of the developed primer sets was evaluated using commercial food products and digested samples with simulated gastric juice. CONCLUSION: All of the developed species-specific primer sets were able to detect target DNA successfully in commercial food products and the digested samples. Therefore, the developed species-specific primer sets in the present study would be useful tools for distinguishing between poisonous plants and edible plants. © 2020 Society of Chemical Industry.

Upregulated immuno-modulator PD-L1 in malignant peripheral nerve sheath tumors provides a potential biomarker and a therapeutic target.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are rare aggressive sarcomas with poor prognosis. More than half of MPNSTs develop from benign precursor tumors associated with neurofibromatosis type 1 (NF1) which is a tumor suppressor gene disorder. Early detection of malignant transformation in NF1 patients is pivotal to improving survival. The primary aim of this study was to evaluate the role of immuno-modulators as candidate biomarkers of malignant transformation in NF1 patients with plexiform neurofibromas as well as predictors of response to immunotherapeutic approaches. METHODS: Sera from a total of 125 NF1 patients with quantified internal tumor load were included, and 25 of them had MPNSTs. A total of six immuno-modulatory factors (IGFBP-1, PD-L1, IFN-α, GM-CSF, PGE-2, and AXL) were measured in these sera using respective ELISA. RESULTS: NF1 patients with MPNSTs had significantly elevated PD-L1 levels in their sera compared to NF1 patients without MPNSTs. By contrast, AXL concentrations were significantly lower in sera of NF1-MPNST patients. IGFBP-1 and PGE2 serum levels did not differ between the two patient groups. IFN-α and GM-CSF were below the detectable level in most samples. CONCLUSION: The immuno-modulator PD-L1 is upregulated in MPNST patients and therefore may provide as a potential biomarker of malignant transformation in patients with NF1 and as a response predictor for immunotherapeutic approaches.

Parallel generation of easily selectable multiple nephronal cell types from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide a source for the generation of defined kidney cells and renal organoids applicable in regenerative medicine, disease modeling, and drug screening. These applications require the provision of hPSC-derived renal cells by reproducible, scalable, and efficient methods. We established a chemically defined protocol by application of Activin A, BMP4, and Retinoic acid followed by GDNF, which steered hPSCs to the renal lineage and resulted in populations of SIX2+/CITED1+ metanephric mesenchyme- (MM) and of HOXB7+/GRHL2+ ureteric bud (UB)-like cells already by 6 days. Transcriptome analysis corroborated that the PSC-derived cell types at day 8 resemble their renal vesicle and ureteric epithelial counterpart in vivo, forming tubular and glomerular renal cells 6 days later. We demonstrate that starting from hPSCs, our in vitro protocol generates a pool of nephrogenic progenitors at the renal vesicle stage, which can be further directed into specialized nephronal cell types including mesangial-, proximal tubular-, distal tubular, collecting duct epithelial cells, and podocyte precursors after 14 days. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, which are applicable for drug testing or cell-based regenerative therapies.

Tangeretin Ameliorates Glucose-Induced Podocyte Injury through Blocking Epithelial to Mesenchymal Transition Caused by Oxidative Stress and Hypoxia.

Podocyte injury inevitably results in leakage of proteins from the glomerular filter and is vital in the pathogenesis of diabetic nephropathy (DN). The underlying mechanisms of podocyte injury facilitate finding of new therapeutic targets for DN treatment and prevention. Tangeretin is an O-polymethoxylated flavone present in citrus peels with anti-inflammatory and antioxidant properties. This study investigated the renoprotective effects of tangeretin on epithelial-to-mesenchymal transition-mediated podocyte injury and fibrosis through oxidative stress and hypoxia caused by hyperglycemia. Mouse podocytes were incubated in media containing 33 mM glucose in the absence and presence of 1-20 µM tangeretin for up to 6 days. The in vivo animal model employed db/db mice orally administrated with 10 mg/kg tangeretin for 8 weeks. Non-toxic tangeretin inhibited glucose-induced expression of the mesenchymal markers of N-cadherin and α-smooth muscle actin in podocytes. However, the reduced induction of the epithelial markers of E-cadherin and P-cadherin was restored by tangeretin in diabetic podocytes. Further, tangeretin enhanced the expression of the podocyte slit diaphragm proteins of nephrin and podocin down-regulated by glucose stimulation. The transmission electron microscopic images revealed that foot process effacement and loss of podocytes occurred in diabetic mouse glomeruli. However, oral administration of 10 mg/kg tangeretin reduced urine albumin excretion and improved foot process effacement of diabetic podocytes through inhibiting loss of slit junction and adherenes junction proteins. Glucose enhanced ROS production and HIF-1α induction in podocytes, leading to induction of oxidative stress and hypoxia. Similarly, in diabetic glomeruli reactive oxygen species (ROS) production and HIF-1α induction were observed. Furthermore, hypoxia-evoking cobalt chloride induced epithelial-to-mesenchymal transition (EMT) process and loss of slit diaphragm proteins and junction proteins in podocytes, which was inhibited by treating submicromolar tangeretin. Collectively, these results demonstrate that tangeretin inhibited podocyte injury and fibrosis through blocking podocyte EMT caused by glucose-induced oxidative stress and hypoxia.

Aesculetin Attenuates Alveolar Injury and Fibrosis Induced by Close Contact of Alveolar Epithelial Cells with Blood-Derived Macrophages via IL-8 Signaling.

Pulmonary fibrosis is a disease in which lung tissues become fibrous and thereby causes severe respiratory disturbances. Various stimuli induce infiltration of macrophages to the respiratory tract, secreting inflammatory cytokines, which subsequently leads to the development of pulmonary fibrosis. Aesculetin, a major component of the sancho tree and chicory, is known to biologically have antioxidant and anti-inflammatory effects. Human alveolar epithelial A549 cells were cultured for 24 h in conditioned media of THP-1 monocyte-derived macrophages (mCM) with 1-20 µM aesculetin. Micromolar aesculetin attenuated the cytotoxicity of mCM containing inflammatory tumor necrosis factor-α (TNF)-α and interleukin (IL)-8 as major cytokines. Aesculetin inhibited alveolar epithelial induction of the mesenchymal markers in mCM-exposed/IL-8-loaded A549 cells (≈47-51% inhibition), while epithelial markers were induced in aesculetin-treated cells subject to mCM/IL-8 (≈1.5-2.3-fold induction). Aesculetin added to mCM-stimulated A549 cells abrogated the collagen production and alveolar epithelial CXC-chemokine receptor 2 (CXCR2) induction. The production of matrix metalloproteinase (MMP) proteins in mCM-loaded A549 cells was reduced by aesculetin (≈52% reduction), in parallel with its increase in tissue inhibitor of metalloproteinases (TIMP) proteins (≈1.8-fold increase). In addition, aesculetin enhanced epithelial induction of tight junction proteins in mCM-/IL-8-exposed cells (≈2.3-2.5-fold induction). The inhalation of polyhexamethylene guanidine (PHMG) in mice accompanied neutrophil predominance in bronchoalveolar lavage fluid (BALF) and macrophage infiltration in alveoli, which was inhibited by orally administrating aesculetin to mice. Treating aesculetin to mice alleviated PHMG-induced IL-8-mediated subepithelial fibrosis and airway barrier disruption. Taken together, aesculetin may antagonize pulmonary fibrosis and alveolar epithelial barrier disruption stimulated by the infiltration of monocyte-derived macrophages, which is typical of PHMG toxicity, involving interaction of IL-8 and CXCR2. Aesculetin maybe a promising agent counteracting macrophage-mediated inflammation-associated pulmonary disorders.