Endothelial Phospholipase C gamma2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy
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ABSTRACT:
PROVIDER: PRJNA755305 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15481404.fastq.gz | Fastqsanger.gz | |||
| SRR15481405.fastq.gz | Fastqsanger.gz | |||
| SRR15481406.fastq.gz | Fastqsanger.gz | |||
| SRR15481407.fastq.gz | Fastqsanger.gz | |||
| SRR15481408.fastq.gz | Fastqsanger.gz |
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Project description:The initial factor in the occurrence, development, and prognosis of cerebral ischemia is vascular dysfunction in the brain, and vascular remodeling of the brain is the key therapeutic target and strategy for ischemic tissue repair. Limb remote ischemic preconditioning exhibits potential pleiotropic protective effects in many brain-related diseases, including stroke.Whether limb remote ischemic preconditioning has other effects such as vascular protective effects and the detailed mechanism by which limb remote ischemic preconditioning improves pathology and angiogenesis in cerebral ischemia remains to be further elucidated. The present study was designed to investigate whether limb remote ischemic preconditioning protects vascular structure and promotes angiogenesis in cerebral ischemic rats.
2024-06-05 | PXD052843 |
Project description:Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.
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Project description:The single cell transcriptional profile from three diabetic and five non-diabetic human skin samples was analyzed The spatial transcriptomics of 1 non-diabetic and 1 diabetic human skin were analyzed
2022-05-04 | GSE182208 | GEO
Project description:Diabetic wounds often suffer from poor perfusion. Reliance on VEGF therapy to improve perfusion makes logical sense, yet clinical study outcomes fall far short of expectations. Our previous work has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. However, the underlying mechanisms remained unknown. Emerging evidence supports the notion that hyperglycemia leads to DNA methylation-based gene silencing. Given that epigenetic changes are reversible, this work guided by scRNA sequencing of human wound-edge, tests the efficacy of gene-targeted therapeutic demethylation with respect to enabling VEGF therapy. Bipedicle ischemic wounds were placed in diabetic mice. PLCγ2 promoter CpG methylation were analyzed using bisulfite sequencing. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR/dCas9-based demethylation cocktail was delivered to the wound-edge using tissue nanotransfection (TNT) technology. The functional outcome of such demethylation was assessed using perfusion imaging. PLCγ2 promoter was hypermethylated at murine diabetic ischemic wound-edge. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with increased abundance of vWF+/PLCγ2+ vascular tissue elements by activating p42/p44-MAPK→HIF1α pathway. Taken together, TNT-based endothelial demethylation of the PLCγ2 gene promoter improved VEGF therapy on the perfusion of cutaneous diabetic wounds resulting in improved closure.
2025-02-25 | GSE268834 | GEO
Project description:Background: Despite improved surgical approaches for chronic limb threatening ischemia (CLTI), amputation rates remain high and contributing tissue-level factors remain unknown.
Objective: The purpose of this study was twofold: 1) to identify differences between the healthy adult and CLTI limb muscle proteome, and 2) to identify differences in the limb muscle proteome of CLTI patients prior to surgical intervention or at the time of amputation.
Methods: Gastrocnemius muscle was collected from non-ischemic control (n=26) and either pre-interventional surgery (n=10) or at amputation outcome (n=29) CLTI patients. All samples were subjected to isobaric tandem-mass-tag-assisted quantitative proteomics.
Results: The mitochondrion was the primary classification of downregulated proteins (>70%) in CLTI limb muscles and paralleled robust functional mitochondrial impairment. Upregulated proteins (>38%) were largely from the extracellular matrix. Across the two independent sites, 39 proteins were downregulated and 20 upregulated uniformly. Pre-interventional CLTI muscles revealed a robust upregulation of mitochondrial proteins but modest functional impairments in fatty acid oxidation as compared with controls. Comparison of pre-intervention and amputation CLTI limb muscles revealed mitochondrial proteome and functional deficits similar to that between amputation and healthy controls. Interestingly, these observed changes occurred despite 50% of the amputation CLTI patients having undergone a prior surgical intervention.
Conclusions: The CLTI proteome supports failing mitochondria as a mechanism contributing to amputation outcomes. The signature of pre-intervention CLTI muscle reveals stable mitochondrial protein abundance that is insufficient to uniformly prevent functional impairments. Taken together, our findings support the investigation of mitochondrial functional therapeutics as both adjuvant and independent therapies for CLTI.
2022-04-21 | PXD025810 | JPOST Repository
Project description:Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intra-cerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth, and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon ligation of the dCLN afferent lymphatics, and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.
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Project description:Using the highly sensitive miRNA array, we screened 411 differentially expressed genes microRNAs in the ischemic muscle with proanthocyanidins treatment and we explored the functions of these miRNAs in the ischemic muslce by Gene Ontology and Kyoto Encyclopedia of Genes annotation. The enrichment results indicated that these miRNAs mainly participated in the muscle regeneration process. Furthermore, the quantitative real-time polymerase chain reaction, immunohistochemistry and dual luciferase reporter assay results showed that miR-133b-3p is a specific regulator of MKP1/ p38-MAPK signal axis, offering a novel therapeutic opportunity for limb ischemic injury.
2023-03-01 | GSE146772 | GEO
Project description:Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intra-cerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth, and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon ligation of the dCLN afferent lymphatics, and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.
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Project description:Peripheral artery disease (PAD) can cause limb pain and ulcers that won't heal. It is a leading cause of amputation, yet the moleculer mechanims that drive disease are poorly understood. To gain insights into mechanims of PAD at the transcriptome level, we performed sc-RNA-seq on non-ischemic and ischemic tissue from a male PAD patient who had undergone a below knee amputation.
2024-08-19 | GSE266788 | GEO
Project description:Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype.
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