Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells.

BACKGROUND: Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window. METHODS: We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry. RESULTS: Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes. CONCLUSIONS: Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

The interaction of estrogen and CSE/H2S pathway in the development of atherosclerosis.

Both estrogen and hydrogen sulfide (H2S) have been shown to inhibit the development of atherosclerosis. We previously reported that cystathionine γ-lyase knockout (CSE-KO) male mice develop atherosclerosis earlier than male wild-type (WT) mice. The present study investigated the interaction of CSE/H2S pathway and estrogen on the development of atherosclerosis in female mice. Plasma estrogen levels were significantly lower in female CSE-KO mice than in female WT mice. NaHS treatment had no effect on plasma estrogen levels in both WT and CSE-KO female mice. After CSE-KO and WT female mice were fed with atherogenic diet for 12 wk, plasma lipid levels were significantly increased and triglyceride levels decreased compared with those of control diet-fed mice. Atherogenic diet induced more atherosclerotic lesion, oxidative stress, intracellular adhesion molecule-1 (ICAM-1), and NF-κB in CSE-KO mice than in WT mice. Estrogen treatment of atherogenic diet-fed WT mice attenuated hypercholesterolemia, oxidative stress, ICAM-1 expression, and NF-κB in WT mice but not in atherogenic diet-fed CSE-KO mice. Furthermore, H2S production in both the liver and vascular tissues was enhanced by estrogen in WT mice but not in CSE-KO mice. It is concluded that the antiatherosclerotic effect of estrogen is mediated by CSE-generated H2S. This study provides new insights into the interaction of H2S and estrogen signaling pathways on the regulation of cardiovascular functions.NEW & NOTEWORTHY Female cystathionine γ-lyase (CSE)-knockout mice have significantly lower plasma estrogen levels and more severe early atherosclerotic lesion than female wild-type mice. H2S production in liver and vascular tissues is enhanced by estrogen via its stimulatory effect on CSE activity. The antiatherosclerotic effect of estrogen is mediated by CSE-generated H2S.

Hydrogen sulfide and the liver.

Hydrogen sulfide (H2S) is a gasotransmitter that regulates numerous physiological and pathophysiological processes in our body. Enzymatic production of H2S is catalyzed by cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST). All these three enzymes present in the liver and via H2S production regulate liver functions. The liver is the hub for metabolism of glucose and lipids, and maintains the level of circulatory lipids through lipoprotein metabolism. Hepatic H2S metabolism affects glucose metabolism, insulin sensitivity, lipoprotein synthesis, mitochondrial biogenetics and biogenesis. Malfunction of hepatic H2S metabolism may be involved in many liver diseases, such as hepatic fibrosis and hepatic cirrhosis.

Decreased endogenous production of hydrogen sulfide accelerates atherosclerosis.

BACKGROUND: Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H2S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H2S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H2S in atherosclerosis development. METHODS AND RESULTS: Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N-acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. CONCLUSIONS: Endogenously synthesized H2S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H2S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H2S pathway is an important therapeutic target for protection against atherosclerosis.

Hydrogen sulfide protects against cellular senescence via S-sulfhydration of Keap1 and activation of Nrf2.

AIMS: H2S, a third member of gasotransmitter family along with nitric oxide and carbon monoxide, exerts a wide range of cellular and molecular actions in our body. Cystathionine gamma-lyase (CSE) is a major H2S-generating enzyme in our body. Aging at the cellular level, known as cellular senescence, can result from increases in oxidative stress. The aim of this study was to investigate how H2S attenuates oxidative stress and delays cellular senescence. RESULTS: Here we showed that mouse embryonic fibroblasts isolated from CSE knockout mice (CSE KO-MEFs) display increased oxidative stress and accelerated cellular senescence in comparison with MEFs from wild-type mice (WT-MEFs). The protein expression of p53 and p21 was significantly increased in KO-MEFs, and knockdown of p53 or p21 reversed CSE deficiency-induced senescence. Incubation of the cells with NaHS (a H2S donor) significantly increased the glutathione (GSH) level and rescued KO-MEFs from senescence. Nrf2 is a master regulator of the antioxidant response, and Keap1 acts as a negative regulator of Nrf2. NaHS S-sulfhydrated Keap1 at cysteine-151, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream genes, such as glutamate-cysteine ligase and GSH reductase. INNOVATION: These results provide a mechanistic insight into how H2S signaling mediates cellular senescence induced by oxidative stress. CONCLUSION: H2S protects against cellular aging via S-sulfhydration of Keap1 and Nrf2 activation in association with oxidative stress.

Interaction of hydrogen sulfide and estrogen on the proliferation of vascular smooth muscle cells.

Hydrogen sulfide (H(2)S) can be endogenously generated from cystathionine gamma-lyase (CSE) in cardiovascular system, offering a cardiovascular protection. It is also known that the lower risk of cardiovascular diseases in female is partially attributed to the protective effect of estrogen. The current study explores the interaction of H(2)S and estrogen on smooth muscle cell (SMC) growth. In the present study, we found that the proliferation of cultured vascular SMCs isolated from wild-type mice (WT-SMCs) was inhibited, but that from CSE gene knockout mice (CSE-KO-SMCs) increased, by estrogen treatments. The expression of estrogen receptor α (ERα), but not ERß, was significantly decreased in CSE-KO-SMCs compared with that in WT-SMCs. Exogenously applied H(2)S markedly increased ERα but not ERß expression. In addition, the inhibition of ER activation and knockdown of ERα expression in WT-SMCs or the overexpression of ERα in CSE-KO-SMCs reversed the respective effects of estrogen on cell proliferation. The expression of cyclin D1 was reduced in WT-SMCs but increased in CSE-KO-SMCs after estrogen treatments, which was reversed by knockdown of ERα in WT-SMCs or overexpression of ERα in CSE-KO-SMCs, respectively. The overexpression of cyclin D1 in WT-SMCs or knockdown of cyclin D1 expression in CSE-KO-SMCs reversed the effects of estrogen on cell proliferation. These results suggest that H(2)S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERα/cyclin D1 pathways.

Integrated stress response modulates cellular redox state via induction of cystathionine γ-lyase: cross-talk between integrated stress response and thiol metabolism.

The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine ß-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4(-/-)) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4(-/-) MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4(-/-) MEFs could be rescued from l-Hcy-induced apoptosis by ß-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine ß-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4(+/+) MEFs. Consistent with ATF4(-/-) MEFs, CSE(-/-) MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE(+/+) MEFs. Liver and kidney GSH levels were also reduced in CSE(-/-) mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.

A critical life-supporting role for cystathionine γ-lyase in the absence of dietary cysteine supply.

This study examined the important relationship between cystathionine γ-lyase (CSE) functionality and cysteine supply for normal growth and life span. Mice with a targeted deletion of the CSE gene (CSE-KO) were fed a cysteine-limited diet and their growth and survival patterns as well as levels of cysteine, homocysteine, glutathione, and hydrogen sulfide (H2S) were measured. CSE-KO mice fed a cysteine-limited diet exhibited growth retardation; decreased levels of cysteine, glutathione, and H2S; and increased plasma homocysteine level. However, histological examinations of liver did not reveal any abnormality and plasma levels of aspartate aminotransferase, alanine aminotransferase, and albumin were normal in these animals. No CSE-KO mice survived after 12 weeks of feeding with the cysteine-limited diet. Supplementation of H2S to the CSE-KO mice failed to reverse the aforementioned abnormalities. On the other hand, supplementation of cysteine in the drinking water of the CSE-KO mice significantly increased plasma cysteine and glutathione levels. This eventually led to an increase in body weight and rescued the animals from death. In conclusion, CSE is critical for cysteine biosynthesis through the transsulfuration pathway and the combination of CSE deficiency and lack of dietary cysteine supply would threaten life sustainability.

Cystathionine gamma-lyase deficiency and overproliferation of smooth muscle cells.

AIMS: Cystathionine gamma-lyase (CSE)-derived H2S plays an important role in regulating cell growth. Lack of CSE expression results in development of hypertension. The current study compared proliferation of smooth muscle cells derived from CSE gene knockout mice (SMCs-KO) with that of wild-type mice (SMCs-WT). METHODS AND RESULTS: Cell proliferation was assessed by bromodeoxyuridine incorporation. Gene expression was analysed by western blotting, real-time PCR, and microarray analysis. Enhanced cell proliferation was detected in SMCs-KO and in the media of the aorta from CSE KO mice. SMCs-KO underwent significantly more apoptosis than SMCs-WT when treated with exogenous H2S (100 microM). CSE KO mice showed much lower level of phosphorylated extracellular signal-regulated kinase (ERK1/2) in mesentery arteries compared with those of WT mice, and exogenous H2S induced more phosphorylation of ERK1/2 in SMCs-KO compared with that in SMCs-WT. Decreased p21(Cip/WAF-1) but increased cyclin D1 expression was observed in isolated SMCs and vascular tissues from CSE KO mice, and exogenous H2S caused more increase in p21(Cip/WAF-1) expression and more decrease in cyclin D1 expression in SMCs-KO than in SMCs-WT. The transcriptional expression of calcitonin receptor-like, intergrin beta 1, and heparin-binding epidermal growth factor-like growth factor was also significantly increased in the aorta of CSE KO mice. CONCLUSION: SMCs-KO display an increased proliferation rate in vitro and in vivo, and these cells are more susceptible to apoptosis induced by exogenous H2S at physiologically relevant concentrations. These cellular effects of H2S are mediated by phosphorylation of ERK1/2 and altered expression of cyclin D1 and p21(Cip/WAF-1).