HHcy Mice Research Articles

Catalpol inhibits HHcy-induced EndMT in endothelial cells by modulating ROS/NF-κB signaling

BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis (AS). Endothelial mesenchymal transition (EndMT) refers to the process in which endothelial cells lose endothelial cell morphology and characteristic gene expression, and acquire phenotypic characteristics and gene expression related to mesenchymal cells. Numerous studies have confirmed that EndMT is involved in the formation of atherosclerosis. Catalpol is one of the active components of Rehmannia, which has antioxidant, anti-inflammatory, anti-tumor, neuroprotective and other biological activities. Studies have shown that catalpol can reduce atherosclerotic plaque induced by high sugar or fat. However, the effect of catalpol on HHCY-induced EndMT is unclear.Methods and resultsIn vitro HHcy-treated primary human umbilical vein endothelial cells (HUVECs) were used to construct a cell model, and the antioxidants N-acetylcysteine (NAC) and catalase alcohol were administered. In vivo C57BL/6N mice were given a diet fed with 4.4% high methionine chow to construct a HHcy mice model and were treated with catalpol. The results showed that hhcy could induce morphological transformation of endothelial cells into mesenchymal cells, increase intracellular ROS content, up-regulate α-SMA, N-cadherin, p-p65 protein expression, down-regulate VE-cadherin, CD31 protein expression, induce pathological changes of aortic root endothelium, and increase aortic endothelial ROS content. Catalpol reversed these hhcy induced outcomes.ConclusionsCatalpol inhibits HHcy-induced EndMT, and the underlying mechanism may be related to the ROS/NF-κB signaling pathway. Catalpol may be a potential drug for the treatment of HHcy-related AS.

Hyperhomocysteinemia Transcriptionally Regulates Expression of a Set of Ion Channels in Brain and Heart Tissues in Mice

The alterations of ion channel gene expressions in brain and heart tissues in HHcy have not been previously reported. We investigated the mRNA expression levels in brain and heart tissues of the HHcy mice compared to the control mice to characterize distinct expression of 36 ion channels. C57BL/6 J. mice were divided into two groups of 15 animals each: (1) control group and (2) HHcy group. The HHcy was induced by methionine administiration. The mRNA levels of ion channels were analyzed using qRT-PCR. TUNEL staining and MDA assay were used for verification of the negative side effects of HHcy in heart and brain tissues. RT-PCR revealed the upregulation of Hcn4, Trpc3, Trpm2 and the downregulation of Abbc8, Cacna1b, Cacna1c, Cacna1e, Cacna1h, Hcn1, Kcnc3, Kcnh7, Kcnj8, Trpc4, Trpc5, Trpc6, Trpm3, Trpm4, Trpv4, Trpv6 in brain tissues of the HHcy group compared to the control. The upregulation of ion channel expressions in heart tissues were not detected, but we found only the downregulation of Accn1, Accn2, Accn3, Hcn1, Kcnc4 and Trpv6 ion channels. Apoptozis and MDA level were significantly increased in brain and heart tissues of the HHcy group compared to the control. Brain tissues compared to heart tissues exhibit a very considerable and diverse ion channel gene expression pattern in mice with HHcy than control. Clarifying the roles of ion channels in HHcy could shed light on the development of novel therapeutic strategies and ultimately improve HHcy side effects.

Excess homocysteine inhibits pancreatic β-cell secretory function by repressing Zbtb20 expression

Homocysteine (Hcy) is a sulfur-containing amino acid. An elevated level of Hcy is a risk factor for diabetes development. However, the mechanism of its effect on pancreatic β-cell function is unclear. In this study, we constructed a hyperhomocysteinemia (HHcy) mouse model by feeding mice a high methionine diet (HMD). The mice suffered impaired glucose tolerance and reduced insulin secretion. Furthermore, at the cellular level, INS1 cells exhibited impaired insulin secretory function after the Hcy intervention. Transcriptomics revealed that Zbtb20 expression was downregulated and the downstream gene Fbp1 was upregulated in HHcy-induced mice compared with mice fed with normal diet. Insulin secretion could be restored by Zbtb20 overexpression or fructose 1,6-bisphosphatase (FBPase) activity inhibition in INS1 cells. In conclusion, our study suggested that Hcy inhibited the insulin secretory function of pancreatic β-cells by suppressing Zbtb20 expression, leading to the development of diabetes. Zbtb20 may be a key target in the development of diabetes associated with elevated Hcy levels.

Homocysteine modulates CXCL10/CXCR3 axis activity to induce endothelial dysfunction.

Elevated homocysteine (Hcy) levels have been linked to the development of cardiovascular diseases, notably endothelial dysfunction, a critical precursor to atherosclerosis. In this extensive investigation, we explore the intricate pathways through which Hcy influences endothelial dysfunction, with particular attention to the CXCL10/CXCR3 axis. Employing a dual approach encompassing both in vitro and in vivo models, we scrutinize the repercussions of Hcy exposure on endothelial functionality. Our results reveal that Hcy significantly impairs crucial endothelial processes, including cell migration, proliferation, and tube formation. Concomitantly, Hcy upregulates the expression of adhesion molecules, exacerbating endothelial dysfunction. In a murine hyperhomocysteinemia (HHcy) model, we observed a parallel increase in plasma Hcy levels and adverse vascular effects. Moreover, our study unraveled a pivotal role of the CXCL10/CXCR3 axis in Hcy-induced endothelial dysfunction. Hcy exposure led to the upregulation of CXCL10 and CXCR3, both in vitro and in HHcy mice. Importantly, the blockade of this axis, achieved through specific antibodies or NBI-74330, mitigated the detrimental effects of Hcy on endothelial function. In conclusion, our findings illuminated the central role of the CXCL10/CXCR3 axis in mediating Hcy-induced endothelial dysfunction, providing valuable insights for potential therapeutic strategies in managing HHcy-related cardiovascular diseases.

Astragaloside Ⅳ negatively regulates Gpr97-TPL2 signaling to protect against hyperhomocysteine-exacerbated sepsis associated acute kidney injury

BackgroundHyperhomocysteine (HHcy) plays an important role in promoting inflammation and cell death of tubular epithelial cells. However, the role of HHcy and Astragaloside IV (AS-IV) in sepsis associated acute kidney injury (S-AKI) remain unclear. PurposeA significant aspect of this study aimed to elucidate the effect of AS-Ⅳ treatment on HHcy-exacerbated S-AKI and reveal its potential mechanism. MethodsMale C57BL/6 J mice fed with specific diet containing 2% methionine were established as in vivo models, and AS-Ⅳ was orally administrated continuously for 3 weeks, and then LPS (10 mg·kg−1 bodyweight) was given by a single intraperitoneal injection. The renal morphological changes were evaluated by HE and PAS staining. RNA-sequencing analysis was applied to select key signaling. The NRK-52E cells exposed to Hcy or combined with LPS were used as in vitro models. The mRNA and protein expression levels of Gpr97-TPL2 signaling were examined by qRT-PCR and western blotting assays. ResultsIn vivo, HHcy mice developed more severe renal injury and prevalent tubular inflammation after LPS injection. In vitro, the levels of NGAL, Gpr97 and TPL2 were significantly increased in NRK-52E cells induced by Hcy (1.6 mM) or in combination with LPS. Notably, the effects of Hcy on TPL2 signaling was abolished by transfecting TPL2 siRNA or treating TPL2 inhibitor, without alterations in Gpr97. However, the enhancement of Gpr97-TPL2 signaling induced by Hcy was counteracted by Gpr97 siRNA. Subsequently, our findings demonstrated that AS-Ⅳ treatment can improve renal function in HHcy-exacerbated S-AKI mice. Mechanistically, AS-Ⅳ alleviated renal tubular damage characterized by abnormal increases in KIM-1, NGAL, TPL2, Gpr97, Sema3A and TNF-α, and decreases in survivin in vivo and in vitro mainly through suppressing the activation of Gpr97-TPL2 signaling. ConclusionThe present study suggested that HHcy-exacerbated S-AKI was mediated mechanically by activation of Gpr97-TPL2 signaling for the first time. Furthermore, our research also illustrated that AS-Ⅳ protected against HHcy-exacerbated S-AKI by attenuating renal tubular epithelial cells damage through negatively regulating Gpr97-TPL2 signaling, proposing a natural product treatment strategy for HHcy-exacerbated S-AKI.

Targeting Reactive Astrocytes in Vascular Dementia: Investigation of Neuronal-Astrocyte-Vascular Interactions.

Historically known as neuronal support cells, astrocytes are now widely studied for their close structural and functional interactions with multiple neural cell types and cerebral vessels where they maintain an ideal environment for optimized brain function. Under pathological conditions, astrocytes become reactive and lose key protective functions. In this commentary, we discuss our recent work in The Journal of Neuroscience (Sompol et al., 2023) that showed Ca2+ dysregulation in reactive astrocytes, as well as hyperactivation of the Ca2+-dependent protein phosphatase calcineurin (CN) and the Nuclear Factor of Activated T Cells (NFATs), in a diet-induced hyperhomocystienemia (HHcy) mouse model of Vascular Contributions to Cognitive Impairment and Dementia (VCID). Intravital multiphoton imaging coupled with whisker stimulation was used to explore astrocyte Ca2+ signaling and neurovascular function under active phase, fully awake conditions. Interestingly, evoked Ca2+ transients in individual astrocytes were greater, even though intercorrelated Ca2+ signaling across networks of astrocytes was impaired in HHcy mice. Blockade of astrocytic CN/NFAT reduced signs of astrocyte reactivity, normalized cerebrovascular function, and improved hippocampal synaptic strength and hippocampal dependent cognition in HHcy mice, revealing a previously unrecognized deficit regarding neuron-astrocyte-vascular interactions. These findings strongly support the use of astrocyte targeting strategies to mitigate pathophysiological changes associated with VCID and other Alzheimer's-related dementias.

Astrocyte Ca2+ dysregulation and neurovascular coupling deficits in a diet‐based mouse model of small cerebral vessel disease

AbstractBackgroundMice exposed to a diet deficient in B6/B12 vitamins and enriched in methionine exhibit hyperhomocysteinemia (HHcy) concomitant with many pathologic features of vascular cognitive impairment and dementia: a very common Alzheimer’s disease related dementia. Here, we evaluated the impact of astrocyte Ca2+ dysregulation and astrocytic Ca2+ signaling pathways in the context of functional hyperemia in fully awake mice exposed to HHcy diet.MethodAdult C57/BL6 mice were fed for 15 weeks with control chow, or chow deficient in vitamins B6/B12 and enriched in methionine (HHcy diet). Spontaneous and evoked (via air puff whisker stimulation) Ca2+ transients in astrocytes of fully awake mice were investigated in barrel cortex using the Ca2+ sensor GCaMP6f (expressed specifically in astrocytes using AAV) and two‐photon imaging. In other experiments, mice were treated with AAV‐Gfa2‐VIVIT to inhibit the Ca2+‐sensitive calcineurin/NFAT pathway in astrocytes, which regulates reactive astrocyte phenotypes. Two‐photon imaging was then used to assess diet and VIVIT effects on functional hyperemia in barrel cortex of fully awake mice.ResultCa2+ transients in individual barrel cortex astrocytes were significantly augmented (greater in amplitude, with faster rise/decay kinetics) in mice treated with HHcy diet. However, the functional connectivity in astrocyte networks of HHcy mice was significantly impaired, particularly during engagement of brain activity with whisker stimulation. Compared to control diet mice, HHcy mice also showed impaired functional hyperemia during whisker stimulation characterized by impaired arteriole dilations and reduced red blood cell velocity in nearby capillaries. Inhibition of reactive astrocyte signaling in HHcy diet mice with VIVIT significantly ameliorated these neurovascular coupling deficits.ConclusionThe results show that astrocyte Ca2+ dysregulation is a major feature of HHcy and contributes to cerebrovascular dysfunction through hyperactivation of the astrocytic CN/NFAT pathway.

Astrocyte signaling regulates microglial and oligodendrocyte subpopulation changes in a diet‐based model of small cerebral vessel disease

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is the second leading cause of dementia behind Alzhiemer’s disease. Hyperhomocysteinemia (HHcy)‐inducing diets in mice recapitulate aspects of VCID including small cerebrovessel pathology, vascular inflammation and cognitive decline. Recently, we found signs of astrocytic Ca2+‐dysregulation and hyperactivation of calcineurin/NFAT activity in mice treated with HHcy diet (Sompol et al., 2023 J Neurosci 43:1797). Inhibition of astrocytic NFATs normalized cerebrovascular, synaptic, and cognitive function in HHcy mice, suggesting that reactive astrocytes alter the brain milieu during development of HHcy and small cerebrovessel pathology. Here, we used scRNA‐seq to further characterize the impact of astrocytic calcineurin/NFATs on multiple brain cell types in the context of HHcy.MethodSeven‐to‐eight week‐old male C57BL/6J mice received intrahippocampal injections of AAV2/5‐Gfa2‐EGFP (control) or AAV expressing the NFAT inhibitor VIVIT (i.e. AAV2/5‐Gfa2‐VIVIT‐EGFP). Mice were then fed with control chow(CT) or HHcy‐inducing chow enriched in methionine and deficient in folate and vitamins B6 and B12. After15 weeks of diet, brains were extracted and hippocampi dissected away, pooled and dissociated for transcriptional analyses. scRNA‐seq was used to identify diet and AAV‐dependent cell specific gene expression changes according to our previous work (Lee et al, 2023, Cell Reports, 112196).Result31 cell clusters were identified and assigned to 1 of 12 unique cell types using established gene markers. Analysis of differentially‐expressed genes showed that microglia and oligodendrocytes were most sensitive to diet and/or AAV treatment. In HHcy mice treated with control AAV (HHcy–EGFP), predominant microglial subpopulations exhibited upregulation of several disease‐associated microglial genes (e.g. Apoe, Il1b, Ccl5, Cebpb, Cybb) and downregulation of common homeostatic genes (e.g. P2ry12, Gpr34, Fcrls, Sparc). Oligodendrocyte subpopulations in the same mice also exhibited upregulation of disease related genes (e.g. Serpina3n, H2‐D1, C4b, Klk6). However, these patterns were mitigated in HHcy mice treated with AAV‐Gfa2‐VIVIT, who exhibited expression patterns in line with CT diet mice. Analyses of DEGs in other cell types and association with immunometabolic pathways are in progress.ConclusionResults suggest that reactive astrocyte signaling strongly regulates molecular phenotypes of other local glial cells and provides a potential target for preventing/resolving pathologic glial activity in VCID and other ADRDs.

Identification of methylation-regulated genes modulating microglial phagocytosis in hyperhomocysteinemia-exacerbated Alzheimer’s disease

BackgroundHyperhomocysteinemia (HHcy) has been linked to development of Alzheimer’s disease (AD) neuropathologically characterized by the accumulation of amyloid β (Aβ). Microglia (MG) play a crucial role in uptake of Aβ fibrils, and its dysfunction worsens AD. However, the effect of HHcy on MG Aβ phagocytosis remains unstudied.MethodsWe isolated MG from the cerebrum of HHcy mice with genetic cystathionine-β-synthase deficiency (Cbs−/−) and performed bulk RNA-seq. We performed meta-analysis over transcriptomes of Cbs−/− mouse MG, human and mouse AD MG, MG Aβ phagocytosis model, human AD methylome, and GWAS AD genes.ResultsHHcy and hypomethylation conditions were identified in Cbs−/− mice. Through Cbs−/− MG transcriptome analysis, 353 MG DEGs were identified. Phagosome formation and integrin signaling pathways were found suppressed in Cbs−/− MG. By analyzing MG transcriptomes from 4 AD patient and 7 mouse AD datasets, 409 human and 777 mouse AD MG DEGs were identified, of which 37 were found common in both species. Through further combinatory analysis with transcriptome from MG Aβ phagocytosis model, we identified 130 functional-validated Aβ phagocytic AD MG DEGs (20 in human AD, 110 in mouse AD), which reflected a compensatory activation of Aβ phagocytosis. Interestingly, we identified 14 human Aβ phagocytic AD MG DEGs which represented impaired MG Aβ phagocytosis in human AD. Finally, through a cascade of meta-analysis of transcriptome of AD MG, functional phagocytosis, HHcy MG, and human AD brain methylome dataset, we identified 5 HHcy-suppressed phagocytic AD MG DEGs (Flt1, Calponin 3, Igf1, Cacna2d4, and Celsr) which were reported to regulate MG/MΦ migration and Aβ phagocytosis.ConclusionsWe established molecular signatures for a compensatory response of Aβ phagocytosis activation in human and mouse AD MG and impaired Aβ phagocytosis in human AD MG. Our discoveries suggested that hypomethylation may modulate HHcy-suppressed MG Aβ phagocytosis in AD.

Atorvastatin rescues hyperhomocysteinemia-induced cognitive deficits and neuroinflammatory gene changes

BackgroundEpidemiological data suggests statins could reduce the risk of dementia, and more specifically, Alzheimer’s disease (AD). Pre-clinical data suggests statins reduce the risk of dementia through their pleiotropic effects rather than their cholesterol lowering effects. While AD is a leading cause of dementia, it is frequently found co-morbidly with cerebral small vessel disease and other vascular contributions to cognitive impairment and dementia (VCID), which are another leading cause of dementia. In this study, we determined if atorvastatin ameliorated hyperhomocysteinemia (HHcy)-induced VCID.MethodsWild-type (C57Bl6/J) mice were placed on a diet to induce HHcy or a control diet each with or without atorvastatin for 14 weeks. Mice underwent novel object recognition testing before tissue collection. Plasma total cholesterol and total homocysteine as well as related metabolites were measured. Using qPCR and NanoString technology, we profiled glial cell-associated gene expression changes. Finally, microglial morphology, astrocyte end feet, and microhemorrhages were analyzed using histological methods.ResultsAtorvastatin treatment of HHcy in mice led to no changes in total cholesterol but decreases in total homocysteine in plasma. While HHcy decreased expression of many glial genes, atorvastatin rescued these gene changes, which mostly occurred in oligodendrocytes and microglia. Microglia in HHcy mice with atorvastatin were trending towards fewer processes compared to control with atorvastatin, but there were no atorvastatin effects on astrocyte end feet. While atorvastatin treatment was trending towards increasing the area of microhemorrhages in HHcy mice in the frontal cortex, it only slightly (non-significantly) reduced the number of microhemorrhages. Finally, atorvastatin treatment in HHcy mice led to improved cognition on the novel object recognition task.ConclusionsThese data suggest that atorvastatin rescued cognitive changes induced by HHcy most likely through lowering plasma total homocysteine and rescuing gene expression changes rather than impacts on vascular integrity or microglial changes.

Hyperhomocysteinemia lowers serum testosterone concentration via impairing testosterone production in Leydig cells.

Hyperhomocysteinemia (HHcy) plays a salient role in male infertility. However, whether HHcy interferes with testosterone production remains inconclusive. Here, we reported a lower serum testosterone level in HHcy mice. Single-cell RNA sequencing revealed that genes related to testosterone biosynthesis, together with nuclear receptor subfamily 5 group A member 1 (Nr5a1), a key transcription factor for steroidogenic genes, were downregulated in the Leydig cells (LCs) of HHcy mice. Mechanistically, Hcy lowered trimethylation of histone H3 on lysine 4 (H3K4me3), which was bound on the promoter region of Nr5a1, resulting in downregulation of Nr5a1. Intriguingly, we identified an unknown cell cluster annotated as Macrophage-like Leydig cells (McLCs), expressing both LCs and macrophages markers. In HHcy mice, McLCs were shifted toward pro-inflammatory phenotype and thus promoted inflammatory response in LC. Betaine supplementation rescued the downregulation of NR5A1 and restored the serum testosterone level in HHcy mice. Overall, our study highlights an etiological role of HHcy in LCs dysfunction.

Hyperhomocysteinemia activates NLRP3 inflammasome to cause hepatic steatosis and insulin resistance via MDM2-mediated ubiquitination of HSF1.

Hyperhomocysteinemia (HHcy) is associated with nonalcoholic fatty liver disease (NAFLD) and insulin resistance (IR). However, the underlying mechanism is still unknown. Recent studies have demonstrated that NLRP3 inflammasome activation plays a vital role in NAFLD and IR. Our study aimed to explore whether NLRP3 inflammasome contributed to HHcy-induced NAFLD and IR as well as dissected the underlying mechanism. C57BL/6 mice were fed a high-methionine diet (HMD) for 8weeks to establish the HHcy mouse model. Compared with a chow diet, HMD induced hepatic steatosis (HS) and IR as well as activation of hepatic NLRP3 inflammasome. Moreover, HHcy-induced NAFLD and IR characterization disclosed that NLRP3 inflammasome activation occurred in liver tissue of HMD-fed mice, but was very marginal in either NLRP3-/- or Caspase-1-/- mice. Mechanistically, high levels of homocysteine (Hcy) up-regulated the expression of mouse double minute 2 homolog (MDM2), which directly ubiquitinates heat shock transcription factor 1 (HSF1) and consequently activated hepatic NLRP3 inflammasome in vivo and in vitro. In addition, in vitro experiments showed P300-mediated HSF1 acetylation at K298 hindered MDM2-mediated ubiquitination of HSF1 at K372, which plays important role in determining the HSF1 level. Importantly, either inhibition of MDM2 by JNJ-165 or activation of HSF1 by HSF1A reversed HMD-induced hepatic NLRP3 inflammasome, and consequently alleviated HS and IR in mice. This study demonstrates that NLRP3 inflammasome activation contributes to HHcy-induced NAFLD and IR, and further identified that HSF1 as a new substrate of MDM2 and its decrease on MDM2-mediated ubiquitination at K372 modulates NLRP3 inflammasome activation. These findings may provide novel therapeutic strategies aimed at halting HS or IR.

Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease.

Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.

Perturbed astrocyte Ca2+ signaling in a diet‐based mouse model of small cerebral vessel disease

AbstractBackgroundThe hyperhomocysteinemia (HHcy) diet model of small cerebral vessel disease exhibits vascular inflammation, cerebral hypoperfusion, impaired neurovascular coupling, synapse dysfunction, and cognitive decline. Pathological changes in astrocyte structural features have also been noted, but functional astrocyte phenotypes have not been characterized. Here, we assessed Ca2+ signaling in individual astrocytes and astrocyte networks in barrel cortex of mice, pretreated with control or HHcy diet. Investigations were performed in fully awake mice to avoid confounding interactions between anesthetics, neural circuit activity, and vasoactive factors.MethodAdult C57/BL6 mice were fed for 15 weeks with control chow, or chow deficient in vitamins B6/B12 and enriched in methionine (HHcy diet). At week 11‐12, mice were injected with AAV‐Gfa104‐lck‐GCaMP6f into barrel cortex, followed by craniotomies and installation of a glass cranial windows with headmounts. Three weeks later, fully awake mice were head‐mounted under a two photon microscope to assess GCaMP6/Ca2+ transient activity in barrel cortex astrocytes during a 10 minute baseline (spontaneous activity) and also before, during, and air puff stimulation of contralateral whiskers (10Hz, 10 sec) . Mean Ca2+ transient amplitudes, transient rise/decay times, and number of transients per region of interest (ROI) were measured and averaged across the field of view (FOV). Functional relationships between multiple ROIs within the FOV were assessed to evaluate astrocyte network activityResultSpontaneous Ca2+ transients were comparable in amplitude and rise/decay kinetics across diet conditions. However, the number of transients per ROI was reduced in HHcy diet mice. Whisker stimulation evoked a primary Ca2+ elevation per ROI followed 10‐15 seconds later by a smaller amplitude Ca2+‐“aftershock”. Evoked Ca2+ signals were highly coordinated across ROIs within the FOV. However, the number of active ROIs and the functional connectivity between ROIs were significantly reduced in HHcy vs. control diet mice. Despite these network deficits, evoked Ca2+ transient amplitudes and aftershocks in individual ROIs were significantly larger with faster activation/decay kinetics in HHcy mice.ConclusionThe results suggest that altered astrocyte Ca2+ signaling, as a result of vascular pathology, may play an important role in the dynamic interplay between cerebral blood flow and neuronal activity, leading to overall neural dysfunction and cognitive impairment.

HHcy Induces Pyroptosis and Atherosclerosis via the Lipid Raft-Mediated NOX-ROS-NLRP3 Inflammasome Pathway in apoE−/− Mice

Lipid rafts play important roles in signal transduction, particularly in responses to inflammatory processes. The current study aimed to identify whether lipid raft-mediated inflammation contributes to hyperhomocysteinemia (HHcy)-accelerated atherosclerosis (AS), and to investigate the underlying mechanisms. THP-1-derived macrophages were used for in vitro experiments. ApoE−/− mice were fed a high-fat diet for 12 weeks to establish an AS model, and a high-fat plus high-methionine diet was used to induce HHcy. We found that homocysteine (Hcy) increased the expression of p22phox and p67phox and promoted their recruitment into lipid rafts (indicating the assembly of the NOX complex), thereby increasing ROS generation and NOX activity, NLRP3 inflammasome activation, and pyroptosis. Mechanistically, Hcy activated the NOX-ROS-NLRP3 inflammasome pathway and induced pyroptosis by increasing the expression of acid sphingomyelinase (ASM) to promote the formation of lipid raft clustering. Importantly, lipid raft-mediated pyroptosis was confirmed in HHcy mice, and HHcy-promoted macrophage recruitment in atherosclerotic lesions and HHcy-aggravated AS were blocked by the lipid raft disruptor methyl-β-cyclodextrin. The study findings indicate that Hcy promotes lipid raft clustering via the upregulation of ASM, which mediates the assembly of the NOX complex, causing an increase in ROS generation, NLRP3 inflammasome activation, and pyroptosis, and contributes to HHcy-induced AS.

Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a.

Endoplasmic reticulum (ER) stress and apoptosis play a critical role in liver injury. Endoplasmic reticulum oxidoreductase 1α (ERO1α) is an oxidase that exists in the luminal side of the ER membrane, participating in protein folding and secretion and inhibiting apoptosis, but the underlying mechanism on liver injury induced by homocysteine (Hcy) remains obscure. In this study, hyperhomocysteinemia (HHcy) mice model was established in cbs +/− mice by feeding a high‐methionine diet for 12 weeks; and cbs +/− mice fed with high‐methionine diet exhibited more severe liver injury compared to cbs +/+ mice. Mechanistically, we found that Hcy promoted ER stress and apoptosis of hepatocytes and thereby aggravated liver injury through inhibiting ERO1α expression; accordingly, overexpression of ERO1α remarkably alleviated ER stress and apoptosis of hepatocytes induced by Hcy. Epigenetic modification analysis revealed that Hcy significantly increased levels of DNA methylation and H3 lysine 9 dimethylation (H3K9me2) on ERO1α promoter, which attributed to upregulated DNA methyltransferase 1 (DNMT1) and G9a, respectively. Further study showed that DNMT1 and G9a cooperatively regulated ERO1α expression in hepatocytes exposed to Hcy. Taken together, our work demonstrates that Hcy activates ER stress and apoptosis of hepatocytes by downregulating ERO1α expression via cooperation between DNMT1 and G9a, which provides new insight into the mechanism of Hcy‐induced ER stress and apoptosis of hepatocytes in liver injury.

Apolipoprotein E ε4/4 genotype limits response to dietary induction of hyperhomocysteinemia and resulting inflammatory signaling

Vascular contributions to cognitive impairment and dementia (VCID) are the second leading cause of dementia behind Alzheimer’s disease. Apolipoprotein E (ApoE) is a lipid transporting lipoprotein found within the brain and periphery. The APOE ε4 allele is the strongest genetic risk factor for late onset Alzheimer’s disease and is a risk factor for VCID. Our lab has previously utilized a dietary model of hyperhomocysteinemia (HHcy) to induce VCID pathology and cognitive deficits in mice. This diet induces perivascular inflammation through cumulative oxidative damage leading to glial mediated inflammation and blood brain barrier breakdown. Here, we examine the impact of ApoE ε4 compared to ε3 alleles on the progression of VCID pathology and inflammation in our dietary model of HHcy. We report a significant resistance to HHcy induction in ε4 mice, accompanied by a number of related differences related to homocysteine (Hcy) metabolism and methylation cycle, or 1-C, metabolites. There were also significant differences in inflammatory profiles between ε3 and ε4 mice, as well as significant reduction in Serpina3n, a serine protease inhibitor associated with ApoE ε4, expression in ε4 HHcy mice relative to ε4 controls. Finally, we find evidence of pervasive sex differences within both genotypes in response to HHcy induction.

Implication of N-Methyl-d-Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-β-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR−/−) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient’s serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.

1150-P: A Hyperhomocysteinemia-Inducing Diet Alters Gut Microbiome and Glucose Homeostasis

Hyperhomocysteinemia (HHcy) is a risk factor for higher mortality in type 2 diabetic patients. A high methionine low folate (HMLF) diet chronically induces HHcy by causing homocysteine (Hcy) accumulation in systemic circulation. A higher systemic Hcy level is clinically associated with the incidence of diabetes mellitus. However, whether HHcy negatively impacts on glucose metabolism and insulin sensitivity remains inconclusive and contradictory. Meanwhile, very few studies have focused on the direct impact of HMLF diet on glucose homeostasis, and that on the gut flora. To understand the impact of HMLF diet, C57BL/6 mice were fed a HMLF diet for 8 consecutive weeks. HMLF diet feeding successfully induced HHcy, caused progressive body weight loss, and slightly impaired glucose homeostasis, as revealed by slight glucose intolerance and insulin resistance, in C57BL/6 mice. Of noted, HMLF diet feeding altered the gut microbiome profile that the relative abundance of porphyromonadaceae family of bacteria was enriched in diet-induced HHcy mice. Interestingly, a previous study correlated the higher frequency of porphyromonadaceae with the development of GLP-1 resistance, implying that HMLF diet might prompt the development of GLP-1 resistance. GLP-1 resistance results in the impairment on glucose-induced insulin secretion. These results provide novel insights into the roles of impaired glucose homeostasis and gut microbiome in the pathogenesis of HHcy-related complications. In short, an improper dietary pattern might impair glucose homeostasis by altering the gut microbiome profile. Disclosure C. Cheng: None. C. Wang: None. Y. Huang: None. Funding Chinese University of Hong Kong (4930709); Hong Kong Research Grants Council Senior Research Fellow Scheme 2020/21 (SRFS2021-4S04)

Hyperhomocysteinemia exacerbates ischemia-reperfusion injury-induced acute kidney injury by mediating oxidative stress, DNA damage, JNK pathway, and apoptosis.

BackgroundHyperhomocysteinemia (HHcy) plays an important role in the progression of many kidney diseases; however, the relationship between HHcy and ischemia-reperfusion injury (IRI)-induced acute kidney injury (IRI-induced AKI) is far from clear. In this study, we try to investigate the effect and possible mechanisms of HHcy on IRI-induced AKI.MethodsTwenty C57/BL6 mice were reared with a regular diet or high methionine diet for 2 weeks (to generate HHcy mice); after that, mice were subgrouped to receive sham operation or ischemia-reperfusion surgery. Twenty four hour after reperfusion, serum creatinine, blood urea nitrogen, and Malondialdehyde (MDA) were measured. H&E staining for tubular injury, western blot for γH2AX, JNK, p-JNK, and cleaved caspase 3, and TUNEL assay for tubular cell apoptosis were also performed.ResultsOur results showed that HHcy did not influence the renal function and histological structure, as well as the levels of MDA, γH2AX, JNK, p-JNK, and tubular cell apoptosis in control mice. However, in IRI-induced AKI mice, HHcy caused severer renal dysfunction and tubular injury, higher levels of oxidative stress, DNA damage, JNK pathway activation, and tubular cell apoptosis.ConclusionOur results demonstrated that HHcy could exacerbate IRI-induced AKI, which may be achieved through promoting oxidative stress, DNA damage, JNK pathway activation, and consequent apoptosis.