Cystathionine Β-synthase Research Articles

Multiplexing Homocysteine into First-Tier Newborn Screening Mass Spectrometry Assays Using Selective Thiol Derivatization.

Classical homocystinuria (HCU) results from deficient cystathionine β-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS. Dried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS). Interferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent-free quantification of tHcy by FIA-MS/MS in first-tier NBS. The combination of tris(2-carboxyethyl)phosphine (TCEP) and NEM yielded significantly less matrix effects compared to dithiothreitol (DTT) and NEM. Analysis of clinical specimens demonstrated that the method could distinguish between HCU-positive, presumptive normal newborns, and newborns receiving total parenteral nutrition. Here we present the first known validated method capable of screening tHcy in DBS during FIA-MS/S first-tier NBS.

Role of Cystathionine β-Synthase and 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation, Migration, and Bioenergetics of Murine Breast Cancer Cells

Cystathionine β-synthase (CBS), CSE (cystathionine γ-lyase) and 3-mercaptopyruvate sulfurtransferase (3-MST) have emerged as three significant sources of hydrogen sulfide (H2S) in various forms of mammalian cancer. Here, we investigated the functional role of CBS' and 3-MST's catalytic activity in the murine breast cancer cell line EO771. The CBS/CSE inhibitor aminooxyacetic acid (AOAA) and the 3-MST inhibitor 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) were used to assess the role of endogenous H2S in the modulation of breast cancer cell proliferation, migration, bioenergetics and viability in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). CBS and 3-MST, as well as expression were detected by Western blotting; H2S production was measured by the fluorescent dye AzMC. The results show that EO771 cells express CBS, CSE and 3-MST protein, as well as several enzymes involved in H2S degradation (SQR, TST, and ETHE1). Pharmacological inhibition of CBS or 3-MST inhibited H2S production, suppressed cellular bioenergetics and attenuated cell proliferation. Cell migration was only inhibited by the 3-MST inhibitor, but not the CBS/CSE inhibitor. Inhibition of CBS/CSE of 3-MST did not significantly affect basal cell viability; inhibition of 3-MST (but not of CBS/CSE) slightly enhanced the cytotoxic effects of oxidative stress (hydrogen peroxide challenge). From these findings, we conclude that endogenous H2S, generated by 3-MST and to a lower degree by CBS/CSE, significantly contributes to the maintenance of bioenergetics, proliferation and migration in murine breast cancer cells and may also exert a minor role as a cytoprotectant.

Role of 3-Mercaptopyruvate Sulfurtransferase (3-MST) in Physiology and Disease.

3-mercaptopyruvate sulfurtransferase (3-MST) plays the important role of producing hydrogen sulfide. Conserved from bacteria to Mammalia, this enzyme is localized in mitochondria as well as the cytoplasm. 3-MST mediates the reaction of 3-mercaptopyruvate with dihydrolipoic acid and thioredoxin to produce hydrogen sulfide. Hydrogen sulfide is also produced through cystathionine beta-synthase and cystathionine gamma-lyase, along with 3-MST, and is known to alleviate a variety of illnesses such as cancer, heart disease, and neurological conditions. The importance of cystathionine beta-synthase and cystathionine gamma-lyase in hydrogen sulfide biogenesis is well-described, but documentation of the 3-MST pathway is limited. This account compiles the current state of knowledge about the role of 3-MST in physiology and pathology. Attempts at targeting the 3-MST pathway for therapeutic benefit are discussed, highlighting the potential of 3-MST as a therapeutic target.

Identification of Key Genes Affecting Flavor Formation in Beijing-You Chicken Meat by Transcriptome and Metabolome Analyses

The flavor of chicken meat is influenced by muscle metabolites and regulatory genes and varies with age. In this study, the metabolomic and transcriptomic data of breast muscle at four developmental stages (days 1, 56, 98, and 120) of Beijing-You chickens (BJYs) were integrated and 310 significantly changed metabolites (SCMs) and 7,225 differentially expressed genes (DEGs) were identified. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SCMs and DEGs were enriched in amino acid, lipid, and inosine monophosphate (IMP) metabolism pathways. Furthermore, genes highly associated with flavor amino acids, lipids, and IMP were identified by a weighted gene co-expression network analysis (WGCNA), including cystathionine β-synthase (CBS), glycine amidinotransferase (GATM), glutamate decarboxylase 2 (GAD2), patatin-like phospholipasedomain containing 6 (PNPLA6), low-specificity L-threonine aldolase (ItaE), and adenylate monophosphate deaminase 1 (AMPD1) genes. A regulatory network related to the accumulation of key flavor components was constructed. In conclusion, this study provides new perspectives regarding the regulatory mechanisms of flavor metabolites in chicken meat during development.

Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells

Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity of endogenously synthesizing cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells’ reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.

H2S Prodrug, SG-1002, Protects against Myocardial Oxidative Damage and Hypertrophy In Vitro via Induction of Cystathionine β-Synthase and Antioxidant Proteins

Endogenously produced hydrogen sulfide (H2S) is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H2S levels have proven cardioprotective in models of acute myocardial infarction (MI) and heart failure (HF). The present study was undertaken to investigate the effects of a novel H2S prodrug, SG-1002, on stress induced hypertrophic signaling in murine HL-1 cardiac muscle cells. Treatment of HL-1 cells with SG-1002 under serum starvation without or with H2O2 increased the levels of H2S, H2S producing enzyme, and cystathionine β-synthase (CBS), as well as antioxidant protein levels, such as super oxide dismutase1 (SOD1) and catalase, and additionally decreased oxidative stress. SG-1002 also decreased the expression of hypertrophic/HF protein markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), galectin-3, TIMP1, collagen type III, and TGF-β1 in stressed HL-1 cells. Treatment with SG-1002 caused a significant induction of cell viability and a marked reduction of cellular cytotoxicity in HL-1 cells under serum starvation incubated without or with H2O2. Experimental results of this study suggest that SG-1002 attenuates myocardial cellular oxidative damage and/or hypertrophic signaling via increasing H2S levels or H2S producing enzymes, CBS, and antioxidant proteins.

Cystathionine Beta-Synthase Deficiency: Three Consecutive Cases Detected in 40 Days by Newborn Screening in Emilia Romagna (Italy) and a Comprehensive Review of the Literature.

Cysthiatonine beta-synthase (CBS) deficiency (CBSD) is an autosomal recessive rare disorder caused by variations on CBS that leads to impaired conversion of homocysteine (Hcy) to cystathionine. Marked hyperhomocysteinemia is the hallmark of the disease. The administration of pyridoxine, the natural cofactor of CBS, may reduce total plasma Hcy. Patient phenotype is classified on pyridoxine responsivity in two groups: pyridoxine-responsive and non-responsive patients. Ectopia lentis, bone deformities, developmental delay, and thromboembolism are the classic signs and symptoms of the disease. Early diagnosis and treatment impact patients' natural history. Therapy aims to lower promptly and maintain Hcy concentrations below 100 μmol/L. Depending on the patient's phenotype, the treatment goals could be obtained by the administration of pyridoxine and/or betaine associated with a methionine-restricted diet. CBSD could be diagnosed in the early days of life by expanded newborn screening (ENS), however, the risk of false negative results is not negligible. In Emilia-Romagna (Italy), during the first 10 years of screening experience, only three cases of CBSD identified have been diagnosed, all in the last two years (incidence 1:118,000 live births). We present the cases and a comprehensive review of the literature to emphasize the role of ENS for early diagnosis of CBSD and its potential pitfalls, reiterating the need for a more effective method to screen for CBSD.

CTH/MPST double ablation results in enhanced vasorelaxation and reduced blood pressure via upregulation of the eNOS/sGC pathway.

Hydrogen sulfide (H2S), a gasotransmitter with protective effects in the cardiovascular system, is endogenously generated by three main enzymatic pathways: cystathionine gamma lyase (CTH), cystathionine beta synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) enzymes. CTH and MPST are the predominant sources of H2S in the heart and blood vessels, exhibiting distinct effects in the cardiovascular system. To better understand the impact of H2S in cardiovascular homeostasis, we generated a double Cth/Mpst knockout (Cth/Mpst -/- ) mouse and characterized its cardiovascular phenotype. CTH/MPST-deficient mice were viable, fertile and exhibited no gross abnormalities. Lack of both CTH and MPST did not affect the levels of CBS and H2S-degrading enzymes in the heart and the aorta. Cth/Mpst -/- mice also exhibited reduced systolic, diastolic and mean arterial blood pressure, and presented normal left ventricular structure and fraction. Aortic ring relaxation in response to exogenously applied H2S was similar between the two genotypes. Interestingly, an enhanced endothelium-dependent relaxation to acetylcholine was observed in mice in which both enzymes were deleted. This paradoxical change was associated with upregulated levels of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) α1 and β1 subunits and increased NO-donor-induced vasorelaxation. Administration of a NOS-inhibitor, increased mean arterial blood pressure to a similar extent in wild-type and Cth/Mpst -/- mice. We conclude that chronic elimination of the two major H2S sources in the cardiovascular system, leads to an adaptive upregulation of eNOS/sGC signaling, revealing novel ways through which H2S affects the NO/cGMP pathway.

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease

Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves.Lower levels of bioavailable H2S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation.We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Transcription factor CgPOU3F4-like regulates expression of pheomelanin synthesis related gene CgB-aat1 in the Pacific oyster (Crassostrea gigas)

Previous study has found that b (0, +) -type amino acid transporter 1 (CgB-aat1) plays an essential role on mantle pigmentation in the Pacific oyster Crassostrea gigas. However, the molecular regulation of CgB-aat1 gene expression remains unclear. Herein, three POU domain family members, CgPOU2F1, CgPOU3F4-like and CgPOU4F3-X1 were characterized and they all had POUs and HOX domains, respectively, which were important in transcriptional regulation. CgPOU3F4-like gene expression was the highest in mantle edge. Subsequently, the dual-luciferase reporter result showed that the core regulatory region of CgB-aat1 gene was from −632 to −350 bp of promoter. In transient co-transfection assays, the strongest activity was activated only by CgPOU3F4-like, suggesting CgPOU3F4-like was a valid transcriptional activator of CgB-aat1 gene promoter. And the structural integrity of CgPOU3F4-like was essential for its activation function. In addition, site directed mutagenesis assay was applied to detect three key binding sites between CgPOU3F4-like and core region of CgB-aat1 gene promoter, and this interaction was verified by ChIP test. Furthermore, CgPOU3F4-like knockdown by RNA interference led to obvious decreases in CgB-aat1 and cystathionine beta-synthase (CgCbs) expressions at both mRNA and protein levels. Collectively, these results indicate that CgPOU3F4-like positively regulate CgB-aat1 gene expression and it may be a critical upstream transcriptional regulation factor in pheomelanin synthesis in C. gigas.

Hypothalamic Paraventricular Nucleus Hydrogen Sulfide Exerts Antihypertensive Effects in Spontaneously Hypertensive Rats via the Nrf2 Pathway.

Hydrogen sulfide (H2S) is widely distributed throughout the nervous system with various antioxidant and anti-inflammatory properties. Hypertension involves an increase in reactive oxygen species (ROS) and inflammation in the hypothalamic paraventricular nucleus (PVN). However, it is unclear how H2S in PVN affects hypertension. Our study used spontaneously hypertensive rats (SHR) and control Wistar Kyoto (WKY) rats, microinjected with adenovirus-associated virus (AAV)-CBS (cystathionine beta-synthase overexpression) or AAV-ZsGreen in bilateral PVN, or simultaneously injected with virus-carrying nuclear factor erythroid 2-related factor 2 (Nrf2)-shRNA for 4 weeks. Blood pressure (BP) and plasma noradrenaline level were detected, and the PVN was collected. Finally, levels of CBS, H2S, Nrf2, Fra-LI, ROS, gp91phox, p47phox, superoxide dismutase 1, interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-α, tyrosine hydroxylase, and glutamate decarboxylase 67 were measured. We found that AAV-CBS increased H2S in the PVN, and BP, neuronal activation, oxidative stress, and inflammation of PVN were substantially reduced. Furthermore, endogenous H2S in the PVN activated Nrf2 and corrected the PVN's imbalance of excitatory and inhibitory neurotransmitters. However, Nrf2 knockdown in the PVN was similarly observed to abolish the beneficial effect of H2S on hypertension. The findings imply that endogenous H2S in SHR PVN is reduced, and PVN endogenous H2S can alleviate hypertension via Nrf2-mediated antioxidant and anti-inflammatory effects.

Current and Novel Therapeutical Approaches of Classical Homocystinuria in Childhood With Special Focus on Enzyme Replacement Therapy, Liver-Directed Therapy and Gene Therapy.

Classical homocystinuria is a hereditary defect of the enzyme cystathionine beta synthase, which is produced in the liver. If this enzyme fails, the synthesis pathway of cysteine from methionine is interrupted, leading to the accumulation of homocysteine in the blood plasma and homocysteine in the urine. After birth, the children are unremarkable except for the characteristic laboratory findings. Symptoms rarely appear before the second year of life. The most common symptom is a prolapse of the crystalline lens. This finding is seen in 70% of untreated 10-year-old affected individuals. As the earliest symptom, psychomotor retardation occurs in the majority of patients already during the first two years of life. Limiting factors in terms of life expectancy are thromboembolism, peripheral arterial disease, myocardial infarction, and stroke. These symptoms are due to the damage to the vessels caused by the elevated amino acid levels. About 30% suffer a thromboembolic event by the age of 20, about half by the age of 30. This review focus on present and new therapeutical approaches like the role of enzyme replacement with presentation of different novel targets in research like pegtibatinase, pegtarviliase, CDX-6512, erymethionase, chaperones, proteasome inhibitors and probiotic treatment with SYNB 1353. Furthermore, we analyze the role of liver-directed therapy with three dimensional (3D) bioprinting, liver bioengineering of liver organoids in vitro and liver transplantation. The role of different gene therapy options to treat and cure this extremely rare disease in childhood will be discussed.

Molecular dynamics characterization of the mechanism of inhibition of ClC-7 activity by PI(3)P.

ClC-7 is a Cl-/H+ antiporter expressed in the lysosome and has been identified as a player in the control of lysosomal pH and ion homeostasis. Known pathologies associated with CLC-7 mutations include osteopetrosis and lysosomal storage diseases. Both pH and current measurements have shown that the signaling lipid PI(3,5)P2 is critical in tuning ClC-7 activity. However, the mechanism by which PI(3,5)P2 achieves this is still unknown. We hypothesized that PI(3,5)P2 stabilizes interactions at the interface between ClC-7's transmembrane domain (TMD) and the cytoplasmic cystathionine beta synthase (CBS) domain, resulting in a TMD-CBS docked conformation that inhibits ClC-7 currents. To test this hypothesis, we have used atomistic molecular dynamics simulations of the transporter in the presence and absence of PI(3)P, a phosphoinositide bound in a recent Cryo-EM structure of CLC-7. Our simulations show that coordination of residues on the CBS with PI(3)P, as well as interactions between residues on the CBS with residues on the TMD play a role in stabilizing the TMD-CBS docked conformation. furthermore, the absence of PI(3)P destabilizes the TMD-CBS docked conformation, increasing the dynamics of the CBS domain. Interestingly, a known disease-causing gain-of-function mutation has similar effects at the TMD-CBS interface. Together our results hint at a mechanism of lipid modulation of ClC-7 activity and pave a way for the development of targeted therapeutics for ClC-7 related pathologies.

ERα/ERβ-directed CBS transcription mediates E2β-stimulated hUAEC H2S production.

Elevated endogenous estrogens stimulate human uterine artery endothelial cell (hUAEC) hydrogen sulfide (H2S) production by selectively upregulating the expression of H2S synthesizing enzyme cystathionine β-synthase (CBS), but the underlying mechanisms are underdetermined. We hypothesized that CBS transcription mediates estrogen-stimulated pregnancy-dependent hUAEC H2S production. Estradiol-17β (E2β) stimulated CBS but not cystathionine γ-lyase (CSE) expression in pregnant human uterine artery ex vivo, which was attenuated by the estrogen receptor (ER) antagonist ICI 182,780. E2β stimulated CBS mRNA/protein and H2S production in primary hUAEC from nonpregnant and pregnant women, but with greater responses in pregnant state; all were blocked by ICI 182,780. Human CBS promoter contains multiple estrogen-responsive elements (EREs), including one ERE preferentially binding ERα (αERE) and three EREs preferentially binding ERβ (βERE), and one full ERE (α/βERE) and one half ERE (½α/βERE) binding both ERα and ERβ. Luciferase assays using reporter genes driven by human CBS promoter with a series of 5'-deletions identified the α/βEREs binding both ERα and ERβ (α/βERE and ½α/βERE) to be important for baseline and E2β-stimulated CBS promoter activation. E2β stimulated ERα/ERβ heterodimerization by recruiting ERα to α/βEREs and βERE, and ERβ to βERE, α/βEREs, and αERE. ERα or ERβ agonist alone trans-activated CBS promoter, stimulated CBS mRNA/protein and H2S production to levels comparable to that of E2β-stimulated, while ERα or ERβ antagonist alone abrogated E2β-stimulated responses. E2β did not change human CSE promoter activity and CSE mRNA/protein in hUAEC. Altogether, estrogen-stimulated pregnancy-dependent hUAEC H2S production occurs by selectively upregulating CBS expression via ERα/ERβ-directed gene transcription.

Cerebroprotective actions of hydrogen sulfide in the epileptic brain in newborn pigs.

Neonatal epileptic seizures cause postictal dysregulation of cerebral blood flow. Hydrogen sulfide (H2S), a mediator with vasodilator and antioxidant properties, is produced in the brain by astrocyte cystathionine β-synthase (CBS). This study investigated whether H2S improves the cerebral vascular outcome of seizures. Epileptic seizures were induced in newborn pigs using bicuculline. The effects of the CBS inhibitor aminooxyacetate (AOA) and the H2S donor NaHS on cerebral vascular outcome of seizures were examined in live pigs, cerebral endothelial cells, and cortical astrocytes. Brain H2S was elevated during seizures. AOA blocked H2S and reduced functional hyperemia in the epileptic brain. The endothelium- and astrocyte-dependent vasodilation of pial arterioles was impaired 48 h after seizures suggesting cerebral vascular dysfunction. Systemic NaHS elevated brain H2S and blocked reactive oxygen species in the epileptic brain and in primary endothelial cells and astrocytes during inflammatory and excitotoxic conditions. Postictal cerebrovascular dysfunction was exaggerated in H2S-inhibited pigs and minimized in NaHS-treated pigs. H2S elevation in the epileptic brain via activation of CBS contributes to functional hyperemia and exhibits cerebroprotective properties. The H2S donor NaHS enhances brain antioxidant defense and provides a therapeutic approach for preventing adverse cerebral vascular outcome of neonatal epileptic seizures. Epileptic seizures in neonates lead to prolonged postictal cerebral vascular dysregulation. The role of hydrogen sulfide (H2S), a mediator with vasodilator and antioxidant properties, in the epileptic brain has been explored. Astrocytes are major sites of enzymatic H2S production in the epileptic brain. Postictal cerebral vascular dysfunction is exaggerated when astrocyte H2S production is pharmacologically inhibited during seizures. Postictal cerebral vascular dysfunction is minimized when the brain H2S is elevated by systemic administration of NaHS during seizures. NaHS provides a therapeutic approach for improving cerebrovascular outcome of epileptic seizures via a mechanism that involves the antioxidant potential of H2S.

Staged, Hybrid Repair Of Bilateral Common Femoral, Profunda, Superficial Femoral, And Popliteal Artery Aneurysms

We report a case of a 69-year-old man with asymptomatic aneurysms of the aorto-ilio-femoropopliteal arteries. CTA showed right side diameters of CFA 3 cm, SFA 2.1 cm, profunda 2.5 cm, popliteal 3.2 cm, and left side CFA 2.6 cm, SFA 2.1 cm, profunda 3.8 cm, popliteal 4.2 cm. Given risk of thrombo-embolic complications, extent of artery disease, and patient preference for limited open surgery, a staged, hybrid approach was utilized. He underwent left-sided bifurcated Dacron interposition graft from the EIA to the SFA and profunda with reimplantation of the largest profunda branch. A Viabahn stent was placed from the SFA anastomosis to P3, sized using IVUS. The right side was addressed seven weeks later with a shorter bifurcated interposition graft from the right EIA to SFA and profunda, with reimplantation of the largest profunda branch. Three branches of the profunda were embolized and a Viabahn stent placed from the profunda anastomosis to a distal target. The popliteal was stented from the SFA anastomosis to P3. Three months post-intervention the left profunda limb thrombosed, though the reimplanted profunda remained patent. He had stable residual endoleaks of the right profunda and bilateral popliteal arteries. He was asymptomatic. Genetic work up showed no pathogenic variants, except for a variant of undetermined significance in the cystathionine beta synthase gene. While optimal management is unclear in such rare pathology, we have found success in short term follow up with a hybrid repair. Patients presenting with this require extensive follow up and multidisciplinary care. View Large Image Figure Viewer Download Hi-res image

Total flavones of Rhododendron induce the transformation of A1/A2 astrocytes via promoting the release of CBS-produced H2S

BackgroundWe previously found that total flavones of Rhododendron (TFR) protected against the cerebral ischemia/reperfusion (I/R) injury. But the detailed mechanism is not clear. Recent research revealed that reactive astrocytes were divided into A1 and A2 phenotypes for their morphological and functional remodeling and neurotoxic- vs-neuroprotective effect on the injury of the central nervous system (CNS). PurposeThe present study was undertaken to explore the role and mechanism of TFR on the phenotypic change of astrocytes following cerebral I/R in vivo and oxygen glucose deprivation/re-oxygenation (OGD/R) in vitro. Study design and methodsWe tested the expression of astrocytes marker glial fibrillary acidic protein (GFAP), A1 astrocytes marker C3 protein and A2 astrocytes marker S100a10, as well as the BrdU/GFAP-positive cells, GFAP/S100a10-positive cells and GFAP/C3-positive cells in mice hippocampal tissues to evaluate the phenotypic change of astrocytes. Besides, we assessed the change of astrocyte phenotypes following OGD/R in vitro. ResultsWe found that mice cerebral I/R promoted the astrocytes proliferation of both A1 and A2 phenotypes in hippocampal tissues. While treatment with TFR could promote the proliferation of A2 astrocytes but inhibit the A1 astrocytes proliferation in mice hippocampal tissues, suggesting that TFR could accelerate the astrocytes transformation into A2 subtype following cerebral I/R. Whereas, in OGD/R model of astrocytes, we found that TFR inhibited the proliferation of both A1 and A2 astrocytes. Besides, we found that TFR could up-regulate the release of cystathionine β-synthase (CBS)-produced hydrogen sulfide (H2S) and inhibit RhoA/Rho kinase pathway, and revealed that the inhibitory effect of TFR on astrocytes proliferation could be blocked by aminooxyacetic acid (AOAA), an CBS inhibitor. Furthermore, TFR could ameliorate the mice cerebral I/R injury and the OGD/R-induced astrocytic damage. ConclusionThese findings suggested that TFR could affect the transformation of astrocytes subtypes following cerebral I/R, which may be related to up-regulation of CBS-produced H2S and subsequent inhibition of RhoA/ROCK pathway.

New insights into the regulation of Cystathionine beta synthase (CBS), an enzyme involved in intellectual deficiency in Down syndrome.

Down syndrome (DS), the most frequent chromosomic aberration, results from the presence of an extra copy of chromosome 21. The identification of genes which overexpression contributes to intellectual disability (ID) in DS is important to understand the pathophysiological mechanisms involved and develop new pharmacological therapies. In particular, gene dosage of Dual specificity tyrosine phosphorylation Regulated Kinase 1A (DYRK1A) and of Cystathionine beta synthase (CBS) are crucial for cognitive function. As these two enzymes have lately been the main targets for therapeutic research on ID, we sought to decipher the genetic relationship between them. We also used a combination of genetic and drug screenings using a cellular model overexpressing CYS4, the homolog of CBS in Saccharomyces cerevisiae, to get further insights into the molecular mechanisms involved in the regulation of CBS activity. We showed that overexpression of YAK1, the homolog of DYRK1A in yeast, increased CYS4 activity whereas GSK3β was identified as a genetic suppressor of CBS. In addition, analysis of the signaling pathways targeted by the drugs identified through the yeast-based pharmacological screening, and confirmed using human HepG2 cells, emphasized the importance of Akt/GSK3β and NF-κB pathways into the regulation of CBS activity and expression. Taken together, these data provide further understanding into the regulation of CBS and in particular into the genetic relationship between DYRK1A and CBS through the Akt/GSK3β and NF-κB pathways, which should help develop more effective therapies to reduce cognitive deficits in people with DS.

Warburg Effect as a Novel Mechanism for Homocysteine-Induced Features of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness. Recent studies have reported impaired glycolysis in AMD patients with a high lactate/pyruvate ratio. Elevated homocysteine (Hcy) (Hyperhomocysteinemia, HHcy) was observed in several clinical studies, reporting an association between HHcy and AMD. We established the effect of HHcy on barrier function, retinal pigment epithelium (RPE) structure, and induced choroidal neovascularization (CNV) in mice. We hypothesize that HHcy contributes to AMD by inducing a metabolic switch in the mitochondria, in which cells predominantly produce energy by the high rate of glycolysis, or "Warburg", effect. Increased glycolysis results in an increased production of lactate, cellular acidity, activation of angiogenesis, RPE barrier dysfunction, and CNV. Evaluation of cellular energy production under HHcy was assessed by seahorse analysis, immunofluorescence, and western blot experiments. The seahorse analysis evaluated the extracellular acidification rate (ECAR) as indicative of glycolysis. HHcy showed a significant increase in ECAR both in vivo using (Cystathionine β-synthase) cbs+/- and cbs-/- mice retinas and in vitro (Hcy-treated ARPE-19) compared to wild-type mice and RPE cells. Moreover, HHcy up-regulated glycolytic enzyme (Glucose transporter-1 (GlUT-1), lactate dehydrogenase (LDH), and hexokinase 1 (HK1)) in Hcy-treated ARPE-19 and primary RPE cells isolated from cbs+/+, cbs+/-, and cbs-/- mice retinas. Inhibition of GLUT-1 or blocking of N-methyl-D-aspartate receptors (NMDAR) reduced glycolysis in Hcy-treated RPE and improved albumin leakage and CNV induction in Hcy-injected mice eyes. The current study suggests that HHcy causes a metabolic switch in the RPE cells from mitochondrial respiration to glycolysis during AMD and confirms the involvement of NMDAR in this process. Therefore, targeting Glycolysis or NMDAR could be a novel therapeutic target for AMD.

One-carbon metabolism and related pathways in ruminal and small intestinal epithelium of lactating dairy cows.

Physiological and environmental stresses such as the transition into lactation and heat load contribute to gastrointestinal tract (GIT) dysfunction. The nonruminant gastrointestinal tract has mechanisms to cope with pro-oxidant and pro-inflammatory stressors arising from the gut lumen or within intestinal cells. One-carbon metabolism (OCM) contributes to anti-oxidant capacity via the production of glutathione (GSH) and taurine, and the synthesis of phospholipid, creatine, and the osmolyte glycinebetaine among others. A multipronged approach was used to assess the biological relevance of OCM and closely-related pathways on GIT function in dairy cows. Ruminal papillae (Rum) and scrapings from duodenum (Duo), jejunum (Jej), and ileum (Ile) were collected at slaughter from eight multiparous Holstein cows averaging 128 ± 12 d in milk and producing 39 ± 5 kg/d. A MIXED model ANOVA with preplanned orthogonal contrasts was used for statistical analysis. Methionine adenosyl transferase 1 activity (MAT) was ~10-fold greater (P < 0.01) and cystathionine β-synthase activity doubled in Rum vs. small intestine. Total glutathione peroxidase (GPX) activity was greatest (P = 0.03) in Ile, but similar to Rum. Activity and mRNA abundance of betaine-homocysteine S-methyltransferase were undetectable. There was a 2.5-fold greater protein abundance of GPX1 (P < 0.01) and a ~2-fold greater abundance of GPX3 (P < 0.01) in Rum vs. small intestine. Among the various amino acids (AA) with roles in OCM or closely-related pathways (e.g. creatine synthesis), concentrations of arginine, aspartate, glutamine, methionine, and serine were lower (P < 0.01) in Rum vs. small intestine. Unlike AA, concentrations of OCM-related intermediates S-5'-adenosyl-homocysteine (SAH), glycinebetaine, carnitine, creatine (CRE), and cysteinesulfinic acid were greater (P < 0.01) while taurine was lower in Rum vs. small intestine. Intermediates of the folate cycle were undetectable. The fact that S-adenosylmethionine (SAM) was undetectable while MAT activity and SAH were greater in Rum suggested that availability of SAM (a methyl donor) is a key determinant of flux through the folate and methionine cycles in the GIT. Except for adenosine, concentrations of glutamate, glycine, α-ketoglutarate, hypotaurine, and GSH were lowest in Ile. Together, the data underscored unique differences in activity of one-carbon metabolism and related pathways across sections of the GIT.