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Restoration of Hydrogen Sulfide Production in Diabetic Mice Improves Reparative Function of Bone Marrow Cells.

ABSTRACT: Bone marrow cell (BMC)-based treatment for critical limb ischemia in diabetic patients yielded a modest therapeutic effect resulting from cell dysfunction. Therefore, approaches that improve diabetic stem/progenitor cell functions may provide therapeutic benefits. Here, we tested the hypothesis that restoration of hydrogen sulfide (H2S) production in diabetic BMCs improves their reparative capacities.Mouse BMCs were isolated by density-gradient centrifugation. Unilateral hind limb ischemia was conducted in 12- to 14-week-old db/+ and db/db mice by ligation of the left femoral artery. The H2S level was measured by either gas chromatography or staining with florescent dye sulfidefluor 7 AM.Both H2S production and cystathionine ?-lyase (CSE), an H2S enzyme, levels were significantly decreased in BMCs from diabetic db/db mice. Administration of H2S donor diallyl trisulfide (DATS) or overexpression of CSE restored H2S production and enhanced cell survival and migratory capacity in high glucose (HG)-treated BMCs. Immediately after hind limb ischemia surgery, the db/+ and db/db mice were administered DATS orally and/or given a local intramuscular injection of green fluorescent protein-labeled BMCs or red fluorescent protein-CSE-overexpressing BMCs (CSE-BMCs). Mice with hind limb ischemia were divided into 6 groups: db/+, db/db, db/db+BMCs, db/db+DATS, db/db+DATS+BMCs, and db/db+CSE-BMCs. DATS and CSE overexpression greatly enhanced diabetic BMC retention in ischemic hind limbs followed by improved blood perfusion, capillary/arteriole density, skeletal muscle architecture, and cell survival and decreased perivascular CD68+ cell infiltration in the ischemic hind limbs of diabetic mice. It is interesting to note that DATS or CSE overexpression rescued high glucose-impaired migration, tube formation, and survival of BMCs or mature human cardiac microvascular endothelial cells. Moreover, DATS restored nitric oxide production and decreased endothelial nitric oxide synthase phosphorylation at threonine 495 levels in human cardiac microvascular endothelial cells and improved BMC angiogenic activity under high glucose condition. Last, silencing CSE by siRNA significantly increased endothelial nitric oxide synthase phosphorylation at threonine 495 levels in human cardiac microvascular endothelial cells.Decreased CSE-mediated H2S bioavailability is an underlying source of BMC dysfunction in diabetes mellitus. Our data indicate that H2S and overexpression of CSE in diabetic BMCs may rescue their dysfunction and open novel avenues for cell-based therapeutics of critical limb ischemia in diabetic patients.

SUBMITTER: Cheng Z

PROVIDER: S-EPMC5101140 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Restoration of Hydrogen Sulfide Production in Diabetic Mice Improves Reparative Function of Bone Marrow Cells.

Cheng Zhongjian Z Garikipati Venkata Naga Srikanth VN Nickoloff Emily E Wang Chunlin C Polhemus David J DJ Zhou Jibin J Benedict Cynthia C Khan Mohsin M Verma Suresh K SK Rabinowitz Joseph E JE Lefer David D Kishore Raj R

Circulation 20160922 19

<h4>Background</h4>Bone marrow cell (BMC)-based treatment for critical limb ischemia in diabetic patients yielded a modest therapeutic effect resulting from cell dysfunction. Therefore, approaches that improve diabetic stem/progenitor cell functions may provide therapeutic benefits. Here, we tested the hypothesis that restoration of hydrogen sulfide (H<sub>2</sub>S) production in diabetic BMCs improves their reparative capacities.<h4>Methods</h4>Mouse BMCs were isolated by density-gradient centr ...[more]

PMID: 27660293

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Project description:IntroductionDiabetes mellitus results in high rates of cardiovascular disease, such as microcirculation disorder of the lower limbs, with angiogenesis impairment being the main factor. The endothelium functions as a barrier between blood and the vessel wall. Vascular endothelial cell dysfunction caused by hyperglycemia is the main factor leading to angiogenesis impairment. Hydrogen sulfide (H2S) and miR-126-3p are known for their pro-angiogenesis effects; however, little is known about how H2S regulates miR-126-3p to promote angiogenesis under high-glucose conditions.ObjectivesThe main objective of this research was to explore how H2S regulates the miR-126-3p levels under high-glucose conditions.MethodsWe evaluated the pro-angiogenesis effects of H2S in the diabetic hindlimb of an ischemia mice model and in vivo Matrigel plugs. Two microRNA datasets were used to screen microRNAs regulated by both diabetes and H2S. The mRNA and protein levels were detected through real-time PCR and Western blot, respectively. Immunofluorescent staining was also used to assess the capillary density and to evaluate the protein levels in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were used in in vitro experiments. A scratch wound-healing assay was applied to detect the migration ability of endothelial cells. Methylated DNA immunoprecipitation combined with real-time PCR was chosen to identify the DNA methylation level in the HUVECs.ResultsExogenous H2S improved angiogenesis in diabetic mice. miR-126-3p was regulated by both diabetes and H2S. Exogenous H2S up-regulated the miR-126-3p level and recovered the migration rate of endothelial cells via down-regulating the DNMT1 protein level, which was increased by high glucose. Furthermore, DNMT1 upregulation in the HUVECs increased the methylation levels of the gene sequences upstream of miR-126-3p and then inhibited the transcription of primary-miR-126, thus decreasing the miR-126-3p level. CSE overexpression in the HUVECs rescued the miR-126-3p level, by decreasing the methylation level to improve migration.ConclusionH2S increases the miR-126-3p level through down-regulating the methylation level, by decreasing the DNMT1 protein level induced by high glucose, thus improving the angiogenesis originally impaired by high glucose.

Project description:Background and purposeHydrogen sulfide (H2 S) is a gaseous signal molecule with antioxidative properties. Sirtuin 3 (SIRT3) is closely associated with mitochondrial function and oxidative stress. The study was to investigate whether and how H2 S improved myocardial hypertrophy via a SIRT3-dependent manner.Experimental approachNeonatal rat cardiomyocytes were pretreated with NaHS (50 μM) for 4 h followed by angiotensin II (Ang II, 100 nM) for 24 h. SIRT3 was silenced with siRNA technology. SIRT3 promoter activity and expression, cell surface, hypertrophic gene mRNA expression, mitochondrial oxygen consumption rate and membrane potential were measured. Male 129S1/SvImJ [wild-type (WT)] and SIRT3 knockout (KO) mice were injected with NaHS (50 μmol·kg-1 ·day-1 ; i.p.) followed by transverse aortic constriction (TAC). Echocardiography, heart mass, mitochondrial ultrastructure, volume and number, oxidative stress, mitochondria fusion and fission-related protein expression were measured.Key resultsIn vitro, NaHS increased SIRT3 promoter activity and SIRT3 expression in Ang II-induced cardiomyocyte hypertrophy. SIRT3 silencing abolished the ability of NaHS to reverse the Ang II-induced cardiomyocyte hypertrophy, mitochondrial function impairment and permeability potential dysfunction, along with the decline in FOXO3a and SOD2 expression. In vivo, after TAC. NaHS attenuated myocardial hypertrophy, inhibited oxidative stress, improved mitochondrial ultrastructure, suppressed mitochondrial volume but increased mitochondrial numbers, enhanced OPA1, MFN1 and MFN2 expression but suppressed DRP1 and FIS1 expression in WT mice but not in SIRT3 KO mice CONCLUSION AND IMPLICATIONS: NaHS improved mitochondrial function and inhibited oxidative stress in myocardial hypertrophy in a SIRT3-dependent manner.Linked articlesThis article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Project description:Fish are a convenient model for the study of reparative and post-traumatic processes of central nervous system (CNS) recovery, because the formation of new cells in their CNS continues throughout life. After a traumatic injury to the cerebellum of juvenile masu salmon, Oncorhynchus masou, the cell composition of the neurogenic zones containing neural stem cells (NSCs)/neural progenitor cells (NPCs) in the acute period (two days post-injury) changes. The presence of neuroepithelial (NE) and radial glial (RG) neuronal precursors located in the dorsal, lateral, and basal zones of the cerebellar body was shown by the immunohistochemical (IHC) labeling of glutamine synthetase (GS). Progenitors of both types are sources of neurons in the cerebellum of juvenile O. masou during constitutive growth, thus, playing an important role in CNS homeostasis and neuronal plasticity during ontogenesis. Precursors with the RG phenotype were found in the same regions of the molecular layer as part of heterogeneous constitutive neurogenic niches. The presence of neuroepithelial and radial glia GS+ cells indicates a certain proportion of embryonic and adult progenitors and, obviously, different contributions of these cells to constitutive and reparative neurogenesis in the acute post-traumatic period. Expression of nestin and vimentin was revealed in neuroepithelial cerebellar progenitors of juvenile O. masou. Patterns of granular expression of these markers were found in neurogenic niches and adjacent areas, which probably indicates the neurotrophic and proneurogenic effects of vimentin and nestin in constitutive and post-traumatic neurogenesis and a high level of constructive metabolism. No expression of vimentin and nestin was detected in the cerebellar RG of juvenile O. masou. Thus, the molecular markers of NSCs/NPCs in the cerebellum of juvenile O. masou are as follows: vimentin, nestin, and glutamine synthetase label NE cells in intact animals and in the post-traumatic period, while GS expression is present in the RG of intact animals and decreases in the acute post-traumatic period. A study of distribution of cystathionine β-synthase (CBS) in the cerebellum of intact young O. masou showed the expression of the marker mainly in type 1 cells, corresponding to NSCs/NCPs for other molecular markers. In the post-traumatic period, the number of CBS+ cells sharply increased, which indicates the involvement of H2S in the post-traumatic response. Induction of CBS in type 3 cells indicates the involvement of H2S in the metabolism of extracellular glutamate in the cerebellum, a decrease in the production of reactive oxygen species, and also arrest of the oxidative stress development, a weakening of the toxic effects of glutamate, and a reduction in excitotoxicity. The obtained results allow us to consider H2S as a biologically active substance, the numerous known effects of which can be supplemented by participation in the processes of constitutive neurogenesis and neuronal regeneration.

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Restoration of Hydrogen Sulfide Production in Diabetic Mice Improves Reparative Function of Bone Marrow Cells.

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Restoration of Hydrogen Sulfide Production in Diabetic Mice Improves Reparative Function of Bone Marrow Cells.

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