Exosomal miR-125b-5p derived from adipose-derived mesenchymal stem cells enhance diabetic hindlimb ischemia repair via targeting alkaline ceramidase 2.

INTRODUCTION: Ischemic diseases caused by diabetes continue to pose a major health challenge and effective treatments are in high demand. Mesenchymal stem cells (MSCs) derived exosomes have aroused broad attention as a cell-free treatment for ischemic diseases. However, the efficacy of exosomes from adipose-derived mesenchymal stem cells (ADSC-Exos) in treating diabetic lower limb ischemic injury remains unclear. METHODS: Exosomes were isolated from ADSCs culture supernatants by differential ultracentrifugation and their effect on C2C12 cells and HUVECs was assessed by EdU, Transwell, and in vitro tube formation assays separately. The recovery of limb function after ADSC-Exos treatment was evaluated by Laser-Doppler perfusion imaging, limb function score, and histological analysis. Subsequently, miRNA sequencing and rescue experiments were performed to figure out the responsible miRNA for the protective role of ADSC-Exos on diabetic hindlimb ischemic injury. Finally, the direct target of miRNA in C2C12 cells was confirmed by bioinformatic analysis and dual-luciferase report gene assay. RESULTS: ADSC-Exos have the potential to promote proliferation and migration of C2C12 cells and to promote HUVECs angiogenesis. In vivo experiments have shown that ADSC-Exos can protect ischemic skeletal muscle, promote the repair of muscle injury, and accelerate vascular regeneration. Combined with bioinformatics analysis, miR-125b-5p may be a key molecule in this process. Transfer of miR-125b-5p into C2C12 cells was able to promote cell proliferation and migration by suppressing ACER2 overexpression. CONCLUSION: The findings revealed that miR-125b-5p derived from ADSC-Exos may play a critical role in ischemic muscle reparation by targeting ACER2. In conclusion, our study may provide new insights into the potential of ADSC-Exos as a treatment option for diabetic lower limb ischemia.

Disruption of adipocyte HIF-1α improves atherosclerosis through the inhibition of ceramide generation.

Atherosclerosis is a chronic multifactorial cardiovascular disease. Western diets have been reported to affect atherosclerosis through regulating adipose function. In high cholesterol diet-fed ApoE -/- mice, adipocyte HIF-1α deficiency or direct inhibition of HIF-1α by the selective pharmacological HIF-1α inhibitor PX-478 alleviates high cholesterol diet-induced atherosclerosis by reducing adipose ceramide generation, which lowers cholesterol levels and reduces inflammatory responses, resulting in improved dyslipidemia and atherogenesis. Smpd3, the gene encoding neutral sphingomyelinase, is identified as a new target gene directly regulated by HIF-1α that is involved in ceramide generation. Injection of lentivirus-SMPD3 in epididymal adipose tissue reverses the decrease in ceramides in adipocytes and eliminates the improvements on atherosclerosis in the adipocyte HIF-1α-deficient mice. Therefore, HIF-1α inhibition may constitute a novel approach to slow atherosclerotic progression.

Sphingolipid Catabolism and Glycerophospholipid Levels Are Altered in Erythrocytes and Plasma from Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system. Changes in lipid metabolism have been suggested to play important roles in MS pathophysiology and progression. In this work we analyzed the lipid composition and sphingolipid-catabolizing enzymes in erythrocytes and plasma from MS patients and healthy controls. We observed reduction of sphingomyelin (SM) and elevation of its products-ceramide (CER) and shingosine (SPH). These changes were supported by the detected up-regulation of the activity of acid sphingomyelinase (ASM) in MS plasma and alkaline ceramidase (ALCER) in erythrocytes from MS patients. In addition, Western blot analysis showed elevated expression of ASM, but not of ALCER. We also compared the ratios between saturated (SAT), unsaturated (UNSAT) and polyunsaturated fatty acids and suggest, based on the significant differences observed for this ratio, that the UNSAT/SAT values could serve as a marker distinguishing erythrocytes and plasma of MS from controls. In conclusion, the application of lipid analysis in the medical practice would contribute to definition of more precise diagnosis, analysis of disease progression, and evaluation of therapeutic strategies. Based on the molecular changes of blood lipids in neurodegenerative pathologies, including MS, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine.

LINC01087 indicates a poor prognosis of glioma patients with preoperative MRI.

Long intergenic non-coding RNA 01,087 (LINC01087) has been concerned as an oncogene in breast cancer, while its mechanism in glioma has been little surveyed. Thus, we searched the prognostic value and functional action of LINC01087 in glioma. Glioma patients after preoperative MRI diagnosis were enrolled, and LINC01087, microRNA (miR)-1277-5p, and alkaline ceramidase 3 (ACER3) levels were tested in glioma cancer tissue. The correlation between LINC01087 expression and the survival of patients were analyzed. LINC01087, miR-1277-5p, and ACER3 levels in U251 cells were altered via transfection, and cell malignant phenotypes were monitored. The relationship between miR-1277-5p and LINC01087 or ACER3 was detected. The LINC01087 and ACER3 expression was in up-regulation and the miR-1277-5p expression was in down-regulation in clinical glioma samples. High expression of LINC01087 was associated with poor prognosis of glioma patients with preoperative MRI. LINC01087 silencing restrained tumor malignancy in glioma cells. Mechanistically, LINC01087 directly interacted with miR-1277-5p. ACER3 was a known target of miR-1277-5p. Moreover, rescue assays reveal that miR-1277-5p overexpression (or ACER3 overexpression) reversed the effects of LINC01087 upregulation (or miR-1277-5p upregulation) on glioma cells. LINC01087 has prognostic significance in glioma and silencing LINC01087 deters glioma development through elevating miR-1277-5p to reduce ACER3 expression.

ACER3-related leukoencephalopathy: expanding the clinical and imaging findings spectrum due to novel variants.

BACKGROUND: Leukodystrophies are the main subgroup of inherited CNS white matter disorders which cause significant mortality and morbidity in early years of life. Diagnosis is mostly based on clinical context and neuroimaging findings; however, genetic tools, particularly whole-exome sequencing (WES), have led to comprehending the causative gene and molecular events contributing to these disorders. Mutation in Alkaline Ceramidase 3 (ACER3) gene which encodes alkaline ceramidase enzyme that plays a crucial role in cellular growth and viability has been stated as an uncommon reason for inherited leukoencephalopathies. Merely only two ACER3 mutations in cases of progressive leukodystrophies have been reported thus far. RESULTS: In the current study, we have identified three novel variants in ACER3 gene in cases with new neurological manifestations including developmental regression, dystonia, and spasticity. The detected variants were segregated into family members. CONCLUSION: Our study expands the clinical, neuroimaging, electroencephalographic, and genetic spectrum of ACER3 mutations. Furthermore, we reviewed and compared the findings of all the previously reported cases and the cases identified here in order to facilitate their diagnosis and management.

Alkaline ceramidase family: The first two decades.

Ceramidases are a group of enzymes that catalyze the hydrolysis of ceramide, dihydroceramide, and phytoceramide into sphingosine (SPH), dihydrosphingosine (DHS), and phytosphingosine (PHS), respectively, along with a free fatty acid. Ceramidases are classified into the acid, neutral, and alkaline ceramidase subtypes according to the pH optima for their catalytic activity. YPC1 and YDC1 were the first alkaline ceramidase genes to be identified and cloned from the yeast Saccharomyces cerevisiae two decades ago. Subsequently, alkaline ceramidase genes were identified from other species, including one Drosophila melanogaster ACER gene (Dacer), one Arabidopsis thaliana ACER gene (AtACER), three Mus musculus ACER genes (Acer1, Acer2, and Acer3), and three Homo sapiens ACER genes (ACER1, ACER2, and ACER3). The protein products of these genes constitute a large protein family, termed the alkaline ceramidase (ACER) family. All the biochemically characterized members of the ACER family are integral membrane proteins with seven transmembrane segments in the Golgi complex or endoplasmic reticulum, and they each have unique substrate specificity. An increasing number of studies suggest that the ACER family has diverse roles in regulating sphingolipid metabolism and biological processes. Here we discuss the discovery of the ACER family, the biochemical properties, structures, and catalytic mechanisms of its members, and its role in regulating sphingolipid metabolism and biological processes in yeast, insects, plants, and mammals.

Human alkaline ceramidase 2 promotes the growth, invasion, and migration of hepatocellular carcinoma cells via sphingomyelin phosphodiesterase acid-like 3B.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. It has a poor prognosis because it is often diagnosed at the advanced stage when treatments are limited. In addition, HCC pathogenesis is not fully understood, and this has affected early diagnosis and treatment of this disease. Human alkaline ceramidase 2 (ACER2), a key enzyme that regulates hydrolysis of cellular ceramides, affects cancer cell survival, however its role in HCC has not been well characterized. Our results showed that ACER2 is overexpressed in HCC tissues and cell lines. In addition, high ACER2 protein expression was associated with tumor growth; ACER2 knockdown resulted in decreased cell growth and migration. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B) promoted HCC cell growth, invasion, and migration; SMPDL3B knockdown had a significant inhibitory effect on HCC tumor growth in vivo. Moreover, ACER2 positively regulated the protein level of SMPDL3B. Of note, ACER2/SMPDL3B promoted ceramide hydrolysis and S1P production. This axis induced HCC survival and could be blocked by inhibition of S1P formation. In conclusion, ACER2 promoted HCC cell survival and migration, possibly via SMPDL3B. Thus, inhibition of ACER2/SMPDL3B may be a novel therapeutic target for HCC treatment.

Alkaline Ceramidase 1 Protects Mice from Premature Hair Loss by Maintaining the Homeostasis of Hair Follicle Stem Cells.

Ceramides and their metabolites are important for the homeostasis of the epidermis, but much remains unknown about the roles of specific pathways of ceramide metabolism in skin biology. With a mouse model deficient in the alkaline ceramidase (Acer1) gene, we demonstrate that ACER1 plays a key role in the homeostasis of the epidermis and its appendages by controlling the metabolism of ceramides. Loss of Acer1 elevated the levels of various ceramides and sphingoid bases in the skin and caused progressive hair loss in mice. Mechanistic studies revealed that loss of Acer1 widened follicular infundibulum and caused progressive loss of hair follicle stem cells (HFSCs) due to reduced survival and stemness. These results suggest that ACER1 plays a key role in maintaining the homeostasis of HFSCs, and thereby the hair follicle structure and function, by regulating the metabolism of ceramides in the epidermis.

Intracellular sphingosine kinase 2-derived sphingosine-1-phosphate mediates epidermal growth factor-induced ezrin-radixin-moesin phosphorylation and cancer cell invasion.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) mediates cellular proliferation, mitogenesis, inflammation, and angiogenesis. These biologies are mediated through S1P binding to specific GPCRs [sphingosine-1-phosphate receptor (S1PR)1-5] and some other less well-characterized intracellular targets. Ezrin-radixin-moesin (ERM) proteins, a family of adaptor molecules linking the cortical actin cytoskeleton to the plasma membrane, are emerging as critical regulators of cancer invasion via regulation of cell morphology and motility. Recently, we identified S1P as an acute ERM activator (via phosphorylation) through its action on S1PR2. In this work, we dissect the mechanism of S1P generation downstream of epidermal growth factor (EGF) leading to ERM phosphorylation and cancer invasion. Using pharmacologic inhibitors, small interfering RNA technologies, and genetic approaches, we demonstrate that sphingosine kinase (SK)2, and not SK1, is essential and sufficient in EGF-mediated ERM phosphorylation in HeLa cells. In fact, knocking down SK2 decreased ERM activation 2.5-fold. Furthermore, we provide evidence that SK2 is necessary to mediate EGF-induced invasion. In addition, overexpressing SK2 causes a 2-fold increase in HeLa cell invasion. Surprisingly, and for the first time, we find that this event, although dependent on S1PR2 activation, does not generate and does not require extracellular S1P secretion, therefore introducing a potential novel model of autocrine/intracrine action of S1P that still involves its GPCRs. These results define new mechanistic insights for EGF-mediated invasion and novel actions of SK2, therefore setting the stage for novel targets in the treatment of growth factor-driven malignancies.

p38 mitogen-activated protein kinase/activator protein-1 involved in serum deprivation-induced human alkaline ceramidase 2 upregulation.

Our previous study revealed that serum deprivation upregulated human alkaline ceramidase 2 (haCER2) activity and mRNA in HeLa cells, but the mechanism remains unknown. In the present study, serum deprivation also upregulated haCER2 activity in HepG2 human hepatoma cell line cells due to an increase in haCER2 mRNA, in which mRNA transcription, not mRNA stability, is involved. Furthermore, p38 mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathway is involved in haCER2 mRNA upregulation by serum deprivation, and this mechanism may explain why haCER2 is upregulated in human liver cancer. In conclusion, p38 MAPK, AP-1 or haCER2 may be used as targets in liver cancer therapy.

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer's Disease.

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer's. Age remains the greatest risk factor for Alzheimer's and the prevalence of age-related late onset Alzheimer's is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer's risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer's and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

Non-genomic rapid responses via progesterone in human peripheral T cells are not indirectly mimicked by sphingosine 1-phosphate.

Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. Progesterone has been shown to induce rapid responses that cause intracellular calcium ([Ca(2+)]i) elevation and acidification followed by inhibition of phytohemagglutinin (PHA)-stimulated proliferation. These rapid responses involve T cell plasma membrane sites, but the mechanisms remain unclear. Three new membrane progesterone receptors (mPRα/mPRß/mPRγ) have been identified as expressed in T cells. These proteins have been identified as G-protein-coupled receptors. Recently, mPRs have been classified as progestin and adipoQ receptors (PAQRs). Furthermore, they have been suggested to be alkaline ceramidases, possibly involved in mediating sphingolipid signaling. Alkaline ceramidases are capable of converting ceramide to sphingosine, which might then be further phosphorylated sphingosine via sphingosine kinase to sphingosine 1-phosphate (S1P). This pathway could result in progesterone acting indirectly via S1P on membrane sphingosine 1-phosphate receptors (S1PRs) in T cells to induce rapid responses. Therefore, our aim was to investigate whether progesterone rapid responses occur indirectly in T cells via S1P. We found that S1P induces [Ca(2+)]i elevation however there was no change in intracellular pH. This is different from the situation with progesterone: S1P alone does not suppress PHA-stimulated cell proliferation and does not act synergistically with progesterone on the inhibition of PHA-induced cell proliferation. In contrast, S1P at 1µM is able to antagonize the proliferation inhibitory effect of progesterone. Thus the rapid responses that are induced by progesterone in human peripheral T cells probably do not involve indirect signaling via S1P and S1PRs.