ERK/Drp1‐dependent mitochondrial fission contributes to HMGB1‐induced autophagy in pulmonary arterial hypertension

ObjectivesHigh‐mobility group box‐1 (HMGB1) and aberrant mitochondrial fission mediated by excessive activation of GTPase dynamin‐related protein 1 (Drp1) have been found to be elevated in patients with pulmonary arterial hypertension (PAH) and critically implicated in PAH pathogenesis. However, it remains unknown whether Drp1‐mediated mitochondrial fission and which downstream targets of mitochondrial fission mediate HMGB1‐induced pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration leading to vascular remodelling in PAH. This study aims to address these issues.MethodsPrimary cultured PASMCs were obtained from male Sprague‐Dawley (SD) rats. We detected RNA levels by qRT‐PCR, protein levels by Western blotting, cell proliferation by Cell Counting Kit‐8 (CCK‐8) and EdU incorporation assays, migration by wound healing and transwell assays. SD rats were injected with monocrotaline (MCT) to establish PAH. Hemodynamic parameters were measured by closed‐chest right heart catheterization.ResultsHMGB1 increased Drp1 phosphorylation and Drp1‐dependent mitochondrial fragmentation through extracellular signal‐regulated kinases 1/2 (ERK1/2) signalling activation, and subsequently triggered autophagy activation, which further led to bone morphogenetic protein receptor 2 (BMPR2) lysosomal degradation and inhibitor of DNA binding 1 (Id1) downregulation, and eventually promoted PASMCs proliferation/migration. Inhibition of ERK1/2 cascade, knockdown of Drp1 or suppression of autophagy restored HMGB1‐induced reductions of BMPR2 and Id1, and diminished HMGB1‐induced PASMCs proliferation/migration. In addition, pharmacological inhibition of HMGB1 by glycyrrhizin, suppression of mitochondrial fission by Mdivi‐1 or blockage of autophagy by chloroquine prevented PAH development in MCT‐induced rats PAH model.ConclusionsHMGB1 promotes PASMCs proliferation/migration and pulmonary vascular remodelling by activating ERK1/2/Drp1/Autophagy/BMPR2/Id1 axis, suggesting that this cascade might be a potential novel target for management of PAH.     

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

Partition of proteins in aqueous two-phase systems containing charged dextran or hydrophobic PEG

Summary The electrochemical effect of a charged dextran derivative and the hydrophobic effect of hydrophobic chain PEG derivative on partitioning of six types of proteins in PEG/dextran aqueous two-phase systems were investigated- When 1. 6%(w/w)DEAE-dextran was present in the system,the partition coefficient decreased quickly with increasing pH value;when 0. 4% (w/w)PEG pentadecanoic acid ester was present in the system, the partition coefficient of protein with strong hydrophobicity was greatly increased. The experimental results show that the influence of hydrocarbon chain PEG derivative on partition coefficient is closely related to the hydrophobicity of proteins.     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

Relationships between Degree of Polymerization and Antioxidant Activities: A Study on Proanthocyanidins from the Leaves of a Medicinal Mangrove Plant Ceriops tagal

Tannins from the leaves of a medicinal mangrove plant, Ceriops tagal, were purified and fractionated on Sephadex LH-20 columns. ¹³C nuclear magnetic resonance (¹³C-NMR), reversed/normal high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDT-TOF MS) analysis showed that the tannins were predominantly B-type procyanidins with minor A-type linkages, galloyl and glucosyl substitutions, and a degree of polymerization (DP) up to 33. Thirteen subfractions of the procyanidins were successfully obtained by a modified fractionation method, and their antioxidant activities were investigated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity and ferric reducing antioxidant power (FRAP) method. All these subfractions exhibited potent antioxidant activities, and eleven of them showed significantly different mean DP (mDP) ranging from 1.43±0.04 to 31.77±1.15. Regression analysis demonstrated that antioxidant activities were positively correlative with mDP when around mDP <10, while dropped and then remained at a level similar to mDP = 5 with around 95 µg ml⁻¹ for DPPH scavenging activity and 4 mmol AAE g⁻¹ for FRAP value.     

Relationship of molecular structure to the mechanism of lysophospholipid-induced smooth muscle cell proliferation

We previously reported that oxidized low-density lipoprotein and one of its constituents, lysophosphatidylcholine (lysoPC), caused smooth muscle cell proliferation that was inhibitable by vitamin E and by a neutralizing antibody against basic fibroblast growth factor-2 (FGF-2). We now show that the mitogenic activity of lysolipids is highly dependent on structure. Phospholipids with palmitoyl fatty acid and phosphocholine induced DNA synthesis optimally. Shorter and longer fatty acids were significantly less potent, as were phosphoserine and phosphoethanolamine head groups. Structurally related phospholipids [platelet-activating factor (PAF) and lysoPAF] were also mitogens and acted via an analogous FGF-2-dependent, vitamin E-inhibitable mechanism. The mechanism of lysoPC stimulation was distinct from that of another phospholipid mitogen, lysophosphatidic acid (lysoPA), in that lysoPC stimulation was not pertussis toxin inhibitable. Furthermore, lysoPA stimulation was not inhibitable by vitamin E. Despite its distinct cellular pathway for stimulation, lysoPA also ultimately led to FGF-2 release. Our data show that specific structural attributes of lysoPC, PAF, and lysoPAF enable these agents to mediate smooth muscle cell release of FGF-2, which in turn stimulates proliferation.     

Zoonotic Trematode Metacercariae in Fish from Phnom Penh and Pursat,Cambodia

A survey was performed to investigate the infection status of freshwater fish with zoonotic trematode metacercariae in Phnom Penh and Pursat Province, Cambodia. All collected fish with ice were transferred to our laboratory and examined using the artificial digestion method. In fish from Phnom Penh, 2 kinds of metacercariae (Opisthorchis viverrini and Haplorchis yokogawai) were detected. O. viverrini metacercariae were positive in 37 (50.0%) of 74 fish in 11 species (average no. metacercariae/fish, 18.6). H. yokogawai metacercariae were detected in 23 (57.5%) of 40 fish in 5 species (average no. metacercariae/fish, 21.0). In fish from Pursat Province, 5 kinds of metacercariae (O. viverrini, H. yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum sp.) were detected; O. viverrini metacercariae (n=3) in 2 fish species (Henicorhynchus lineatus and Puntioplites falcifer), H. yokogawai metacercariae (n=51) in 1 species (P. falcifer), H. pumilio metacercariae (n=476) in 2 species (H. lineatus and Pristolepis fasciata), C. formosanus metacercariae (n=1) in 1 species (H. lineatus), and Procerovum sp. metacercariae (n=63) in 1 species (Anabas testudineus). From the above results, it has been confirmed that various freshwater fish play the role of a second intermediate host for zoonotic trematodes (O. viverrini, H. yokogawai, H. pumilio, C. formosanus, and Procerovum sp.) in Cambodia.     

Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.