Critical role of VGLL4 in the regulation of chronic normobaric hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension (PH), a rare but deadly cardiopulmonary disorder, is characterized by extensive remodeling of pulmonary arteries resulting from enhancement of pulmonary artery smooth muscle cell proliferation and suppressed apoptosis; however, the underlying pathophysiological mechanisms remain largely unknown. Recently, epigenetics has gained increasing prominence in the development of PH. We aimed to investigate the role of vestigial-like family member 4 (VGLL4) in chronic normobaric hypoxia (CNH)-induced PH and to address whether it is associated with epigenetic regulation. The rodent model of PH was established by CNH treatment (10% O2 , 23 hours/day). Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, immunoprecipitation, and adeno-associated virus tests were performed to explore the potential mechanisms involved in CNH-induced PH in mice. VGLL4 expression was upregulated and correlated with CNH in PH mouse lung tissues in a time-dependent manner. VGLL4 colocalized with α-smooth muscle actin in cultured pulmonary arterial smooth muscle cells (PASMCs), and VGLL4 immunoactivity was increased in PASMCs following hypoxia exposure in vitro. VGLL4 knockdown attenuated CNH-induced PH and pulmonary artery remodeling by blunting signal transducer and activator of transcription 3 (STAT3) signaling; conversely, VGLL4 overexpression exacerbated the development of PH. CNH enhanced the acetylation of VGLL4 and increased the interaction of ac-H3K9/VGLL4 and ac-H3K9/STAT3 in the lung tissues, and levels of ac-H3K9, p-STAT3/STAT3, and proliferation-associated protein levels were markedly up-regulated, whereas apoptosis-related protein levels were significantly downregulated, in the lung tissues of mice with CNH-induced PH. Notably, abrogation of VGLL4 acetylation reversed CNH-induced PH and pulmonary artery remodeling and suppressed STAT3 signaling. Finally, STAT3 knockdown alleviated CNH-induced PH. In conclusion, VGLL4 acetylation upregulation could contribute to CNH-induced PH and pulmonary artery remodeling via STAT3 signaling, and abrogation of VGLL4 acetylation reversed CNH-induced PH. Pharmacological or genetic deletion of VGLL4 might be a potential target for therapeutic interventions in CNH-induced PH.

Forskolin induced remodeling of lipid droplets in rat adipocytes.

Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 µmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.

Spontaneous hypertension occurs with adipose tissue dysfunction in perilipin-1 null mice.

Perilipin-1 (Plin1) coats lipid droplets exclusively in adipocytes and regulates two principle functions of adipose tissue, triglyceride storage and hydrolysis, which are disrupted upon Plin1 deficiency. In the present study, we investigated the alterations in systemic metabolites and hormones, vascular function and adipose function in spontaneous hypertensive mice lacking perilipin-1 (Plin1-/-). Plin1-/- mice developed spontaneous hypertension without obvious alterations in systemic metabolites and hormones. Plin1 expressed only in adipose cells but not in vascular cells, so its ablation would have no direct effect in situ on blood vessels. Instead, Plin1-/- mice showed dysfunctions of perivascular adipose tissue (PVAT), a fat depot that anatomically surrounds systemic arteries and has an anticontractile effect. In Plin1-/- mice, aortic and mesenteric PVAT were reduced in mass and adipocyte derived relaxing factor secretion, but increased in basal lipolysis, angiotensin II secretion, macrophage infiltration and oxidative stress. Such multiple culprits impaired the anticontractile effect of PVAT to promote vasoconstriction of aortic and mesenteric arteries of Plin1-/- mice. Furthermore, arterial vessels of Plin1-/- mice showed increasing angiotensin II receptor type 1, monocyte chemotactic protein-1 and interlukin-6 expression, structural damage of endothelial and smooth muscle cells, along with impaired endothelium-dependent relaxation. Hypertension in Plin1-/- mice might occur as a deleterious consequence of PVAT dysfunction. This finding provides the direct evidence that links dysfunctional PVAT to vascular dysfunction and hypertension, particularly in pathophysiological states. This hypertensive mouse model might mimic and explain the hypertension occurring in patients with adipose tissue dysfunction, particularly with Plin1 mutations.

Fascia Origin of Adipose Cells.

Adipocytes might arise from vascular stromal cells, pericytes and endothelia within adipose tissue or from bone marrow cells resident in nonadipose tissue. Here, we identified adipose precursor cells resident in fascia, an uninterrupted sheet of connective tissue that extends throughout the body. The cells and fragments of superficial fascia from the rat hindlimb were highly capable of spontaneous and induced adipogenic differentiation but not myogenic and osteogenic differentiation. Fascial preadipocytes expressed multiple markers of adipogenic progenitors, similar to subcutaneous adipose-derived stromal cells (ASCs) but discriminative from visceral ASCs. Such preadipocytes resided in fascial vasculature and were physiologically active in vivo. In growing rats, adipocytes dynamically arose from the adventitia to form a thin adipose layer in the fascia. Later, some adipocytes appeared to overlay on top of other adipocytes, an early sign for the formation of three-dimensional adipose tissue in fascia. The primitive adipose lobules extended invariably along blood vessels toward the distal fascia areas. At the lobule front, nascent capillaries wrapped and passed ahead of mature adipocytes to form the distal neovasculature niche, which might replenish the pool of preadipocytes and supply nutrients and hormones necessary for continuous adipogenesis. Our findings suggest a novel model for the origin of adipocytes from the fascia, which explains both neogenesis and expansion of adipose tissue. Fascial preadipocytes generate adipose cells to form primitive adipose lobules in superficial fascia, a subcutaneous nonadipose tissue. With continuous adipogenesis, these primitive adipose lobules newly formed in superficial fascia may be the rudiment of subcutaneous adipose tissue. Stem Cells 2016;34:1407-1419.

Hydrogen sulfide ameliorates lipopolysaccharide-induced anxiety-like behavior by inhibiting checkpoint kinase 1 activation in the hippocampus of mice.

Hydrogen sulfide (H2S), an endogenous gasotransmitter, exhibits the anxiolytic roles through its anti-inflammatory effects, although its underlying mechanisms remain largely elusive. Emerging evidence has documented that cell cycle checkpoint kinase 1 (Chk1)-regulated DNA damage plays an important role in the neurodegenerative diseases; however, there are few relevant reports on the research of Chk1 in neuropsychiatric diseases. Here, we aimed to investigate the regulatory role of H2S on Chk1 in lipopolysaccharide (LPS)-induced anxiety-like behavior focusing on inflammasome activation in the hippocampus. Cystathionine γ-lyase (CSE, a H2S-producing enzyme) knockout (CSE-/-) mice displayed anxiety-like behavior and activation of inflammasome-mediated inflammatory responses, manifesting by the increase levels of interleukin-1ß (IL-1ß), IL-6, and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) expression in the hippocampus. Importantly, expression of p-Chk1 and γ-H2AX (DNA damage marker) levels were also increased in the hippocampus of CSE-/- mice. LPS treatment decreased the expression of CSE and CBS while increased p-Chk1 and γ-H2AX levels and inflammasome-activated neuroinflammation in the hippocampus of mice. Moreover, p-Chk1 and γ-H2AX protein levels and cellular immunoactivity were significantly increased while CSE and CBS were markedly decreased in cultured BV2 cells followed by LPS treatment. Treatment of mice with GYY4137, a donor of H2S, inhibited LPS-induced increased in p-Chk1 and γ-H2AX levels, mitigated inflammasome activation and inflammatory responses as well as amelioration of anxiety-like behavior. Notably, SB-218078, a selective Chk1 inhibitor treatment attenuated the effect of LPS on inflammasome activation and inflammatory responses and the induction of anxiety-like behavior. Finally, STAT3 knockdown with AAV-STAT3 shRNA alleviated LPS-induced anxiety-like behavior and inhibited inflammasome activation in the hippocampus, and blockade of NLRP3 with MCC950 attenuated neuroinflammation induction and ameliorated LPS-induced anxiety-like behavior. Overall, this study indicates that downregulation of Chk1 activity by H2S activation may be considered as a valid strategy for preventing the progression of LPS-induced anxiety-like behavior.

Role of Ero1α in cognitive impairment induced by chronic hypoxia.

Recent reports suggested the endoplasmic reticulum stress (ERS)-associated pathway is involved with cognitive impairment in hypoxia condition. ERO1-like protein alpha (Ero1α), an endoplasmic reticulum membrane-bound N-glycoprotein, has been reported to promote oxidative protein folding. However, no studies have reported whether the Ero1α is trapped in hypoxia-induced neuronal loss through the ERS-associated pathways. In our study, this effect of Ero1α was investigated using C57BL/6J mice, the HT22 cells and primary rat neurons. C57BL/6J mice were modeled in a hypoxic chamber for 4 weeks. Behavioral tests were then carried out to test cognitive functions, including the Morris water maze and fear conditioning test. Proteomics showed that Ero1α distinctly upregulated compared with normoxia group and verified using western blotting. Flow cytometry and immunofluorescence were used to analyze the neuroprotective effect of inhibitor EN460 of Ero1α in the HT22 cells. In C57BL/6J mice, hypoxia significantly caused cognitive decline. Brain slice staining results were also used to confirm this effect. Western blot analysis demonstrated that Ero1α, ERS-associated proteins and apoptosis-associated proteins significantly increased in the hypoxia treated groups, further proliferation-related marker protein decreased. EN460, a selective endoplasmic reticulum oxidation 1 (ERO1) inhibitor, counteracted neuronal apoptosis and ameliorated neuronal cell proliferation in the HT22 cells. Taken together, our data indicate that hypoxia induces cognitive impairment, at least in part, by upregulating Ero1α which contributes to neuronal apoptosis through ERS signaling pathway, providing preliminary experimental evidence that the Ero1α is a promising therapeutic target in hypoxia-induced cognitive deficits.

Vascular smooth muscle cell-derived hydrogen sulfide promotes atherosclerotic plaque stability via TFEB (transcription factor EB)-mediated autophagy.

Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H2S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H2S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2+ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68+ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = -0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H2S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H2S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H2S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H2S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H2S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H2S may act as a warning biomarker for vulnerable plaque.Abbreviations ATG9A: autophagy related 9A; CTH: cystathionine gamma-lyase; CQ: chloroquine; HASMCs: human aortic smooth muscle cells; H2S: hydrogen sulfide; LAMP1: lysosomal associated membrane protein 1; LAPTM5: lysosomal protein transmembrane 5; NaHS: sodium hydrosulfide hydrate; ox-LDL: oxidized-low density lipoprotein; PPG: DL- propagylglycine; TFEB: transcription factor EB; 3-MA: 3-methyladenine; VSMCs: vascular smooth muscle cells.

Norswertianolin Promotes Cystathionine γ-Lyase Activity and Attenuates Renal Ischemia/Reperfusion Injury and Hypertension.

Cystathionine gamma-lyase (CSE)/hydrogen sulfide (H2S) plays a protective role in cardiovascular diseases including hypertension and ischemia/reperfusion (I/R) injury. This study was aimed to screen natural small molecule compounds that activate CSE activity and then evaluate its effect(s) on kidney I/R injury and hypertension. Applying computer molecular docking technology, we screened the natural small molecule compound norswertianolin (NW)-specific binding to CSE. Using the microscale thermophoresis technology, we confirmed that the Leu68 site was the essential hydrogen bond site of NW binding to CSE. NW supplementation significantly increased CSE expression and its activity for H2S generation both in vivo and in vitro. In the model of acute and long-term kidney I/R injury, NW pretreatment dramatically attenuated kidney damage, associated with decreasing blood urea nitrogen (BUN), serum creatinine (Cr) level, reactive oxygen species (ROS) production, and cleaved caspase 3 expression. In spontaneously hypertensive rats (SHRs), NW treatment also lowered blood pressure, the media/lumen ratio of the femoral artery, and the mRNA level of inflammatory cytokines. In conclusion, NW acts as a novel small molecular chemical compound CSE agonist, directly binding to CSE, heightening CSE generation-H2S activity, and then alleviating kidney I/R injury and hypertension. NW has a potential therapeutic merit for cardiovascular diseases.

Upregulation of KDM6B contributes to lipopolysaccharide-induced anxiety-like behavior via modulation of VGLL4 in mice.

Histone H3K27me3 demethylase KDM6B (also known as Jumonji domain-containing protein D3, JMJD3) plays vital roles in the etiology of inflammatory responses; however, little is known about the role of KDM6B in neuroinflammation-induced anxiety-like behavior. The present study aimed to investigate the potential role of KDM6B in lipopolysaccharide (LPS)-induced anxiety-like behavior and to evaluate whether it is associated with the modulation of vestigial-like family member 4 (VGLL4). The elevated plus maze, light-dark box, and open-field test were performed to test the anxiety-like behavior induced by LPS in C57BL/6 J male mice. Levels of relative protein expression in the hippocampus were quantified by western blotting. KDM6B inhibitor GSK-J4 and microglia inhibitor minocycline as well as adeno-associated virus of Vgll4 shRNA were used to explore the underlying mechanisms. We found that KDM6B, VGLL4, interleukin-1ß (IL-1ß), and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) protein levels were increased in LPS-dose dependent manner in the hippocampus but not in prefrontal cortex. GSK-J4 treatment attenuated LPS-induced VGLL4, the signal transducer and activator of transcription 3 (STAT3), IL-1ß and Iba-1 upregulation and anxiety-like behavior. Knockdown VGLL4 with Vgll4 shRNA prevented the increase of anxiety-like behavior and levels of STAT3, IL-1ß, and Iba-1 expression in the hippocampus of LPS-treated mice. Moreover, minocycline, an inhibitor of microglia treatment blunted LPS-induced anxiety-like behavior. Collectively, these results demonstrate that the induction of neuroinflammation by LPS promotes KDM6B activation in the hippocampus, and LPS-induced anxiety-like behavior is associated with upregulation of VGLL4 by KDM6B in the hippocampus.

Sulfhydrated Sirtuin-1 Increasing Its Deacetylation Activity Is an Essential Epigenetics Mechanism of Anti-Atherogenesis by Hydrogen Sulfide.

Aims: Hydrogen sulfide (H2S) has a protective role in the pathogenesis of atherosclerosis by multiple pathways. Sirtuin-1 (SIRT1) is a histone deacetylase, as an essential mediated longevity gene, and has an anti-atherogenic effect by regulating the acetylation of some functional proteins. Whether SIRT1 is involved in protecting H2S in atherosclerosis and its mechanism remains unclear. Results: In ApoE-knockout atherosclerosis mice, treatment with an H2S donor (NaHS or GYY4137) reduced atherosclerotic plaque area, macrophage infiltration, aortic inflammation, and plasma lipid level. H2S treatment increased aorta and liver SIRT1 mRNA expression. Overexpression or slicing cystathionine gamma lyase (CSE) also changed intracellular SIRT1 expression. CSE/H2S treatment increased SIRT1 deacetylation in endothelium and hepatocytes and macrophages, then induced deacetylation of its target proteins (P53, P65, and sterol response element binding protein), thereby reducing endothelial and macrophage inflammation and inhibiting macrophage cholesterol uptake and cholesterol de novo synthesis of liver. Also, CSE/H2S induced SIRT1 sulfhydration at its two zinc finger domains, increased its zinc ion binding activity to stabilize the alpha-helix structure, lowered its ubiquitination, and reduced its degradation. Innovation: H2S is a novel SIRT1 activator by direct sulfhydration. Because SIRT1 has a role in longevity, H2S may be a protector for aging-related diseases. Conclusion: Endogenous CSE/H2S directly sulfhydrated SIRT1, enhanced SIRT1 binding to zinc ion, then promoted its deacetylation activity, and increased SIRT1 stability, thus reducing atherosclerotic plaque formation.

Author Correction: Clopidogrel as a donor probe and thioenol derivatives as flexible promoieties for enabling H2S biomedicine.

The original version of this Article contained an error in the spelling of the author Bin Geng, which was incorrectly given as Bing Geng. This has been corrected in both the PDF and HTML versions of the Article.

Clopidogrel as a donor probe and thioenol derivatives as flexible promoieties for enabling H2S biomedicine.

Hydrogen sulfide has emerged as a critical endogenous signaling transmitter and a potentially versatile therapeutic agent. The key challenges in this field include the lack of approved hydrogen sulfide-releasing probes for in human exploration and the lack of controllable hydrogen sulfide promoieties that can be flexibly installed for therapeutics development. Here we report the identification of the widely used antithrombotic drug clopidogrel as a clinical hydrogen sulfide donor. Clopidogrel is metabolized in patients to form a circulating metabolite that contains a thioenol substructure, which is found to undergo spontaneous degradation to release hydrogen sulfide. Model studies demonstrate that thioenol derivatives are a class of controllable promoieties that can be conveniently installed on a minimal structure of ketone with an α-hydrogen. These results can provide chemical tools for advancing hydrogen sulfide biomedical research as well as developing hydrogen sulfide-releasing drugs.

Proteomic analysis of murine testes lipid droplets.

Testicular Leydig cells contain abundant cytoplasmic lipid droplets (LDs) as a cholesteryl-ester store for releasing cholesterols as the precursor substrate for testosterone biosynthesis. Here, we identified the protein composition of testicular LDs purified from adult mice by using mass spectrometry and immunodetection. Among 337 proteins identified, 144 were previously detected in LD proteomes; 44 were confirmed by microscopy. Testicular LDs contained multiple Rab GTPases, chaperones, and proteins involved in glucuronidation, ubiquination and transport, many known to modulate LD formation and LD-related cellular functions. In particular, testicular LDs contained many members of both the perilipin family and classical lipase/esterase superfamily assembled predominately in adipocyte LDs. Thus, testicular LDs might be regulated similar to adipocyte LDs. Remarkably, testicular LDs contained a large number of classical enzymes for biosynthesis and metabolism of cholesterol and hormonal steroids, so steroidogenic reactions might occur on testicular LDs or the steroidogenic enzymes and products could be transferred through testicular LDs. These characteristics differ from the LDs in most other types of cells, so testicular LDs could be an active organelle functionally involved in steroidogenesis.