Use of mouse primary epidermal organoids for USA300 infection modeling and drug screening.

Skin infections caused by drug-resistant Staphylococcus aureus occur at high rates nationwide. Mouse primary epidermal organoids (mPEOs) possess stratified histological and morphological characteristics of epidermis and are highly similar to their derived tissue at the transcriptomic and proteomic levels. Herein, the susceptibility of mPEOs to methicillin-resistant S. aureus USA300 infection was investigated. The results show that mPEOs support USA300 colonization and invasion, exhibiting swollen epithelial squamous cells with nuclear necrosis and secreting inflammatory factors such as IL-1ß. Meanwhile mPEOs beneficial to observe the process of USA300 colonization with increasing infection time, and USA300 induces mPEOs to undergo pyroptosis and autophagy. In addition, we performed a drug screen for the mPEO infection model and showed that vancomycin restores cell viability and inhibits bacterial internalization in a concentration-dependent manner. In conclusion, we establish an in vitro skin infection model that contributes to the examination of drug screening strategies and antimicrobial drug mechanisms.

Overexpression of REST Causes Neuronal Injury and Decreases Cofilin Phosphorylation in Mice.

BACKGROUND: RE1-silencing transcription factor (REST) is known to silence target genes involved in synaptic plasticity and neuronal differentiation. Although previous studies implicate REST in neurodegenerative diseases, its function in the progression of Alzheimer's disease (AD) is uncertain. OBJECTIVE: The aim of the present work was to explore the mechanisms of AD and determine whether and how REST was involved in the pathogenesis of AD. METHODS: We investigated the differentially expressed genes and key transcription factors in AD using bioinformatics analysis. In addition, we assessed the expression of REST under the influence of AD-related factors. Mice overexpressing REST were generated and analyzed by proteomics analysis. We used transmission electron microscopy, Golgi-cox staining, immunohistochemistry, and western blotting to examine the impact of REST on neurons. RESULTS: The results of bioinformatics analysis revealed REST as a hub transcriptional regulator in AD. We demonstrate that the mRNA expression of REST was significantly upregulated compared with that in the control groups, not only in AD patients but also in APP/PS1 transgenic mice, lipopolysaccharide-induced neuroinflammatory mice, and oxidative and glutamate stressed neurons. Using proteomics analysis, we showed that the upregulation of REST increased the expression of genes involved in apoptotic and mitochondrial pathways. Long-term overexpression of REST significantly reduced the number of dendritic spines and increased the mitochondrial defect and apoptosis. Reduction of the cofilin phosphorylation may be one of its mechanisms, and cofilin activity could be affected through the P38 MAPK/CREB signaling pathway. CONCLUSION: These results demonstrated the possible mechanism underlying AD and indicated REST as a potential therapeutic target for AD.

Dietary Geranylgeranyl Pyrophosphate Counteracts the Benefits of Statin Therapy in Experimental Pulmonary Hypertension.

BACKGROUND: The mevalonate pathway generates endogenous cholesterol and intermediates including geranylgeranyl pyrophosphate (GGPP). By reducing GGPP production, statins exert pleiotropic or cholesterol-independent effects. The potential regulation of GGPP homeostasis through dietary intake and the interaction with concomitant statin therapy is unknown. METHODS: We developed a sensitive high-pressure liquid chromatography technique to quantify dietary GGPP and conducted proteomics, qualitative real-time polymerase chain reaction screening, and Western blot to determine signaling cascades, gene expression, protein-protein interaction, and protein membrane trafficking in wild-type and transgenic rats. RESULTS: GGPP contents were highly variable depending on food source that differentially regulated blood GGPP levels in rats. Diets containing intermediate and high GGPP reduced or abolished the effects of statins in rats with hypoxia- and monocrotaline-induced pulmonary hypertension: this was rescuable by methyl-allylthiosulfinate and methyl-allylthiosulfinate-rich garlic extracts. In human pulmonary artery smooth muscle cells treated with statins, hypoxia activated RhoA in an extracellular GGPP-dependent manner. Hypoxia-induced ROCK2 (Rho associated coiled-coil containing protein kinase 2)/Rab10 (Ras-related protein rab-10) signaling was prevented by statin and recovered by exogenous GGPP. The hypoxia-activated RhoA/ROCK2 pathway in rat and human pulmonary artery smooth muscle cells upregulated the expression of Ca2+-sensing receptor (CaSR) and HIMF (hypoxia-induced mitogenic factor), a mechanism attenuated by statin treatment and regained with exogenous GGPP. Rab10 knockdown almost abrogated hypoxia-promoted CaSR membrane trafficking, a process diminished by statin and resumed by exogenous GGPP. Hypoxia-induced pulmonary hypertension was reduced in rats with CaSR mutated at the binding motif of HIMF and the interaction between dietary GGPP and statin efficiency was abolished. In humans fed a high GGPP diet, blood GGPP levels were increased. This abolished statin-lowering effects on plasma GGPP, and also on hypoxia-enhanced RhoA activity of blood monocytes that was rescued by garlic extracts. CONCLUSIONS: There is important dietary regulation of GGPP levels that interferes with the effects of statin therapy in experimental pulmonary hypertension. These observations rely on a key and central role of RhoA-ROCK2 cascade activation and Rab10-faciliated CaSR membrane trafficking with subsequent overexpression and binding of HIMF to CaSR. These findings warrant clinical investigation for the treatment of pulmonary hypertension and perhaps other diseases by combining statin with garlic-derived methyl-allylthiosulfinate or garlic extracts and thus circumventing dietary GGPP variations.

Phenylalanine induces pulmonary hypertension through calcium-sensing receptor activation.

Phenylalanine levels are associated with pulmonary hypertension in metabolic profiling clinical studies. However, the pathophysiological role of phenylalanine on pulmonary circulation is still unclear. We experimentally addressed the direct impact of phenylalanine on pulmonary circulation in rats and explored the underlying molecular pathway. Phenylalanine was injected intraperitoneally into Sprague-Dawley rats (400 mg/100 g body wt) as a single dose or daily in a chronic manner for 2, 3, and 4 wk. Chronic injection of phenylalanine induced pulmonary hypertension with time-dependent severity, evidenced by elevated pulmonary artery pressure and pulmonary vascular resistance as well as pulmonary artery and right ventricular hypertrophy. Using tandem mass spectrometry analysis, we found a quick twofold increase in blood level of phenylalanine 2 h following injection. This increase led to a significant accumulation of phenylalanine in lung after 4 h, which remained sustained at up to a threefold increase after 4 wk. In addition, a cellular thermal shift assay with lung tissues from phenylalanine-injected rats revealed the binding of phenylalanine to the calcium-sensing receptor (CaSR). In vitro experiments with cultured pulmonary arterial smooth muscle cells showed that phenylalanine activated CaSR, as indicated by an increase in intracellular calcium content, which was attenuated or diminished by the inhibition or knockdown of CaSR. Finally, the global knockout or lung-specific knockdown of CaSR significantly attenuated phenylalanine-induced pulmonary hypertension. Chronic phenylalanine injection induces pulmonary hypertension through binding to CaSR and its subsequent activation. Here, we demonstrate a pathophysiological role of phenylalanine in pulmonary hypertension through the CaSR. This study provides a novel animal model for pulmonary hypertension and reveals a potentially clinically significant role for this metabolite in human pulmonary hypertension as a marker, a mediator of disease, and a possible therapeutic target.

Spermine on Endothelial Extracellular Vesicles Mediates Smoking-Induced Pulmonary Hypertension Partially Through Calcium-Sensing Receptor.

Objective- This study aims to determine whether and how the enriched metabolites of endothelial extracellular vesicles (eEVs) are critical for cigarette smoke-induced direct injury of endothelial cells and the development of pulmonary hypertension, rarely explored in contrast to long-investigated mechanisms secondary to chronic hypoxemia. Approach and Results- Metabonomic screen of eEVs from cigarette-smoking human subjects reveals prominent elevation of spermine-a polyamine metabolite with potent agonist activity for the extracellular CaSR (calcium-sensing receptor). CaSR inhibition with the negative allosteric modulator Calhex231 or CaSR knockdown attenuates cigarette smoke-induced pulmonary hypertension in rats without emphysematous changes in lungs or chronic hypoxemia. Cigarette smoke exposure increases the generation of spermine-positive eEVs and their spermine content. Immunocytochemical staining and immunogold electron microscopy recognize the spermine enrichment not only within the cytosol but also on the outer surface of eEV membrane. The repression of spermine synthesis, the inhibitory analog of spermine, N1-dansyl-spermine, Calhex231, or CaSR knockdown profoundly suppresses eEV exposure-mobilized cytosolic calcium signaling, pulmonary artery constriction, and smooth muscle cell proliferation. Confocal imaging of immunohistochemical staining demonstrates the migration of spermine-positive eEVs from endothelium into smooth muscle cells in pulmonary arteries of cigarette smoke-exposed rats. The repression of spermine synthesis or CaSR knockout results in attenuated development of pulmonary hypertension induced by an intravascular administration of eEVs. Conclusions- Cigarette smoke enhances eEV generation with spermine enrichment at their outer surface and cytosol, which activates CaSR and subsequently causes smooth muscle cell constriction and proliferation, therefore, directly leading to the development of pulmonary hypertension.

Hypoxia-Induced Mitogenic Factor Acts as a Nonclassical Ligand of Calcium-Sensing Receptor, Therapeutically Exploitable for Intermittent Hypoxia-Induced Pulmonary Hypertension.

Hypoxia-induced mitogenic factor (HIMF) is an inflammatory cytokine playing important role(s) in the development of hypoxic pulmonary hypertension. The molecular target mediating HIMF-stimulated downstream events remains unclear. The coimmunoprecipitation screen identified extracellular calcium-sensing receptor (CaSR) as the binding partner for HIMF in pulmonary artery smooth muscle cells. The yeast 2-hybrid assay then revealed the binding of HIMF to the intracellular, not the extracellular, domain of extracellular CaSR. The binding of HIMF enhanced the activity of extracellular CaSR and mediated hypoxia-evoked proliferation of pulmonary artery smooth cells and the development of pulmonary vascular remodeling and pulmonary hypertension, all of which was specifically attenuated by a synthesized membrane-permeable peptide flanking the core amino acids of the intracellular binding domain of extracellular CaSR. Thus, HIMF induces pulmonary hypertension as a nonclassical ligand of extracellular CaSR, and the binding motif of extracellular CaSR can be therapeutically exploitable.

Mitochondrial transplantation attenuates hypoxic pulmonary hypertension.

Mitochondria are essential for the onset of hypoxia-induced pulmonary vasoconstriction and pulmonary vascular-remodeling, two major aspects underlying the development of pulmonary hypertension, an incurable disease. However, hypoxia induces relaxation of systemic arteries such as femoral arteries and mitochondrial heterogeneity controls the distinct responses of pulmonary versus femoral artery smooth muscle cells to hypoxia in vitro. The aim of this study was to determine whether mitochondrial heterogeneity can be experimentally exploited in vivo for a potential treatment against pulmonary hypertension. The intact mitochondria were transplanted into Sprague-Dawley rat pulmonary artery smooth muscle cells in vivo via intravenous administration. The immune-florescent staining and ultrastructural examinations on pulmonary arteries confirmed the intracellular distribution of exogenous mitochondria and revealed the possible mitochondrial transfer from pulmonary artery endothelial cells into smooth muscle cells in part through their intercellular space and intercellular junctions. The transplantation of mitochondria derived from femoral artery smooth muscle cells inhibited acute hypoxia-triggered pulmonary vasoconstriction, attenuated chronic hypoxia-induced pulmonary vascular remodeling, and thus prevented the development of pulmonary hypertension or cured the established pulmonary hypertension in rats exposed to chronic hypoxia. Our findings suggest that mitochondrial transplantation possesses potential implications for exploring a novel therapeutic and preventive strategy against pulmonary hypertension.