ABSTRACT
The retina is one of the highest metabolically active tissues with a high oxygen consumption, so insufficient blood supply leads to visual impairment. The incidence of related conditions is increasing; however, no effective treatment without side effects is available. Furthermore, the pathomechanism of these diseases is not fully understood. Our aim was to develop an optimal ischemic retinopathy mouse model to investigate the retinal damage in a time-dependent manner. Retinal ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 10, 13, 15 or 20 min, or by right permanent unilateral common carotid artery occlusion (UCCAO). Optical coherence tomography was used to follow the changes in retinal thickness 3, 7, 14, 21 and 28 days after surgery. The number of ganglion cells was evaluated in the central and peripheral regions on whole-mount retina preparations. Expression of glial fibrillary acidic protein (GFAP) was analyzed with immunohistochemistry and Western blot. Retinal degeneration and ganglion cell loss was observed in multiple groups. Our results suggest that the 20 min BCCAO is a good model to investigate the consequences of ischemia and reperfusion in the retina in a time-dependent manner, while the UCCAO causes more severe damage in a short time, so it can be used for testing new drugs.
Subject(s)
Disease Models, Animal , Glial Fibrillary Acidic Protein , Hypoxia , Ischemia , Retina , Tomography, Optical Coherence , Animals , Mice , Ischemia/metabolism , Ischemia/pathology , Glial Fibrillary Acidic Protein/metabolism , Retina/metabolism , Retina/pathology , Hypoxia/metabolism , Retinal Diseases/metabolism , Retinal Diseases/pathology , Retinal Diseases/etiology , Male , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/metabolism , Mice, Inbred C57BL , Time FactorsABSTRACT
MicroRNAs (miRs) regulate physiological and pathological processes, including ischemia-induced angiogenesis and neovascularization. They can be transferred between cells by extracellular vesicles (EVs). However, the specific miRs that are packaged in EVs released from skeletal muscles, and how this process is modulated by ischemia, remain to be determined. We used a mouse model of hindlimb ischemia and next generation sequencing (NGS) to perform a complete profiling of miR expression and determine the effect of ischemia in skeletal muscles, and in EVs of different sizes (microvesicles (MVs) and exosomes) released from these muscles. Ischemia significantly modulated miR expression in whole muscles and EVs, increasing the levels of several miRs that can have pro-angiogenic effects (angiomiRs). We found that specific angiomiRs are selectively enriched in MVs and/or exosomes in response to ischemia. In silico approaches indicate that these miRs modulate pathways that play key roles in angiogenesis and neovascularization, including HIF1/VEGF signaling, regulation of actin cytoskeleton and focal adhesion, NOTCH, PI3K/AKT, RAS/MAPK, JAK/STAT, TGFb/SMAD signaling and the NO/cGMP/PKG pathway. Thus, we show for the first time that angiomiRs are selectively enriched in MVs and exosomes released from ischemic muscles. These angiomiRs could be targeted in order to improve the angiogenic function of EVs for potential novel therapeutic applications in patients with severe ischemic vascular diseases.
Subject(s)
Extracellular Vesicles , Ischemia , MicroRNAs , Muscle, Skeletal , Neovascularization, Physiologic , Animals , Muscle, Skeletal/metabolism , Muscle, Skeletal/blood supply , Muscle, Skeletal/pathology , Extracellular Vesicles/metabolism , MicroRNAs/metabolism , MicroRNAs/genetics , Ischemia/metabolism , Ischemia/pathology , Mice , Hindlimb/blood supply , Hindlimb/pathology , Mice, Inbred C57BL , Signal Transduction , Male , Exosomes/metabolism , Neovascularization, Pathologic/metabolism , AngiogenesisABSTRACT
Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target.
Subject(s)
Calcium Signaling , Endothelial Cells , Epithelial-Mesenchymal Transition , Mitochondria , RNA-Seq , Single-Cell Analysis , Animals , Humans , Mitochondria/metabolism , RNA-Seq/methods , Mice , Endothelial Cells/metabolism , Epithelial-Mesenchymal Transition/genetics , Calcium Channels/metabolism , Calcium Channels/genetics , Ischemia/metabolism , Ischemia/pathology , Calcium/metabolism , Single-Cell Gene Expression AnalysisABSTRACT
Neuroactive steroids (NASs) directly affect neuronal excitability. Despite their role in the nervous system is intimately linked to pain control, knowledge is currently limited. This study investigates the peripheral involvement of NASs in chronic ischemic pain by targeting the cytochrome P450 side-chain cleavage enzyme (P450scc). Using a rat model of hind limb thrombus-induced ischemic pain (TIIP), we observed an increase in P450scc expression in the ischemic hind paw skin. Inhibiting P450scc with intraplantar aminoglutethimide (AMG) administration from post-operative day 0 to 3 significantly reduced the development of mechanical allodynia. However, AMG administration from post-operative day 3 to 6 did not affect established mechanical allodynia. In addition, we explored the role of the peripheral sigma-1 receptor (Sig-1R) by co-administering PRE-084 (PRE), a Sig-1R agonist, with AMG. PRE reversed the analgesic effects of AMG during the induction phase. These findings indicate that inhibiting steroidogenesis with AMG alleviates peripheral ischemic pain during the induction phase via Sig-1Rs.
Subject(s)
Disease Models, Animal , Hyperalgesia , Ischemia , Rats, Sprague-Dawley , Receptors, sigma , Animals , Hyperalgesia/drug therapy , Hyperalgesia/pathology , Hyperalgesia/complications , Male , Ischemia/complications , Ischemia/pathology , Receptors, sigma/antagonists & inhibitors , Receptors, sigma/metabolism , Sigma-1 Receptor , Pain/drug therapy , Pain/complications , Pain/etiology , Pain/pathology , Hindlimb/drug effects , Rats , Cytochrome P-450 Enzyme System/metabolismABSTRACT
We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of ßIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies.
Subject(s)
Disease Models, Animal , Hindlimb , Ischemia , Muscle, Skeletal , Animals , Rats , Muscle, Skeletal/pathology , Muscle, Skeletal/metabolism , Muscle, Skeletal/blood supply , Hindlimb/blood supply , Hindlimb/pathology , Ischemia/pathology , Ischemia/metabolism , Ischemia/physiopathology , Male , Rats, Wistar , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Tubulin/metabolism , Peripheral Arterial Disease/pathology , Peripheral Arterial Disease/metabolism , Peripheral Arterial Disease/physiopathologyABSTRACT
Acute limb ischemia (ALI) is defined as a sudden reduction in blood flow to a limb, resulting in cessation of blood flow and, therefore, cessation of the delivery of nutrients and oxygen to the tissues of the lower limb. Despite optimal treatment to restore blood flow to ischemic tissues, some patients may suffer from ischemia/reperfusion (I/R) syndrome, the most severe complication after a revascularization procedure used to restore blood flow. There are multiple molecular and cellular factors that are involved in each phase of ALI. This review focuses firstly on molecular and cellular factors of arterial thrombosis, highlighting the role of atherosclerotic plaques, smooth muscle cells (SMCs), and cytokine which may alter key components of the extracellular matrix (ECM). Then, molecular and cellular factors of arterial embolism will be discussed, highlighting the importance of thrombi composition. Molecular and cellular factors of ischemia/reperfusion syndrome are analyzed in depth, highlighting several important mechanisms related to tissue damage, such as inflammation, apoptosis, autophagy, necrosis, and necroptosis. Furthermore, local and general complications of ALI are discussed in the context of molecular alterations. Ultimately, the role of novel biomarkers and targeted therapies is discussed.
Subject(s)
Ischemia , Humans , Ischemia/metabolism , Ischemia/pathology , Animals , Thrombosis/metabolism , Thrombosis/pathology , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Acute Disease , Extremities/blood supply , Extremities/pathologyABSTRACT
"Cysts of the ligamentum flavum (cysts-LF)" is the term for non-neoplastic cystic lesion involving LF. The aim of the present study was to elucidate the histopathological characteristics and pathogenesis of "cysts-LF". Herein, we defined cysts-LF as spinal cysts containing degenerative LF components. From archival cases, we investigated 18 symptomatic cysts-LF surgically removed from 18 patients (13 males and five females; median age 68.5 years [range, 42-86 years]). The elastic fibers of LF components in the wall were separated and/or torn, and cyst walls were accompanied by chondroid metaplasia (17 cases), myxoid changes (13 cases), ossification (11 cases), amyloid deposits (14 cases), hemosiderosis (six cases), granular/smudgy calcification (four cases), synovial cell linings (three cases), and severe inflammatory infiltrates (one case). These histologic features of our cysts-LF were shared by previously reported "cysts-LF." Fourteen cysts-LF demonstrated vascular stenosis/occlusion, and eight showed thick hyalinized vessels, suggesting local circulatory insufficiency. Eight cases (44%) exhibited lipomembranous fat necrosis, accompanied by hyalinized vascular changes (p = 0.003). Ischemic conditions were observed in nearly half of the present cysts-LF, and may be one of the main contributing factors for the formation of cysts-LF, via degeneration and cystic changes in the LF.
Subject(s)
Cysts , Ligamentum Flavum , Humans , Female , Male , Aged , Ligamentum Flavum/pathology , Middle Aged , Adult , Aged, 80 and over , Cysts/pathology , Ischemia/pathologyABSTRACT
Aim: This study aims to investigate the effects of large extracellular vesicles (EVs) induced by pluripotent stem cell-derived mesenchymal stem cells on lower limb ischemic disease and explore its potential mechanisms. Materials & methods: The pathology of muscles was accessed by H&E staining and immunofluorescence staining. In vitro, we conducted wound-healing assay, tube formation assay, RT qPCR, ELISA, RNA sequencing and proteomic analysis. Results: iMSCs-lEVs alleviated the injury of ischemic lower limb and promoted the recovery of lower limb function. In vitro, iMSCs-lEVs promoted the proliferation, migration, and angiogenesis of HMEC-1 cells by regulating the ERK/MAPK signing pathway. Conclusion: This study demonstrated that iMSCs-lEVs promoted endothelial cell angiogenesis via the ERK/MAPK signaling pathway, thereby improving function after lower limb ischemic injury.
[Box: see text].
Subject(s)
Extracellular Vesicles , Induced Pluripotent Stem Cells , Ischemia , MAP Kinase Signaling System , Neovascularization, Physiologic , Extracellular Vesicles/metabolism , Animals , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Ischemia/therapy , Ischemia/metabolism , Ischemia/pathology , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Mice , Cell Proliferation , Lower Extremity/blood supply , Cell Movement , Male , AngiogenesisABSTRACT
BACKGROUND: This study explores the potential role of Thioredoxin-interacting protein (TXNIP) silencing in endothelial colony-forming cells (ECFCs) within the scope of age-related comorbidities and impaired vascular repair. We aim to elucidate the effects of TXNIP silencing on vasculogenic properties, paracrine secretion, and neutrophil recruitment under conditions of metabolic stress. METHODS: ECFCs, isolated from human blood cord, were transfected with TXNIP siRNA and exposed to a high glucose and ß-hydroxybutyrate (BHB) medium to simulate metabolic stress. We evaluated the effects of TXNIP silencing on ECFCs' functional and secretory responses under these conditions. Assessments included analyses of gene and protein expression profiles, vasculogenic properties, cytokine secretion and neutrophil recruitment both in vitro and in vivo. The in vivo effects were examined using a murine model of hindlimb ischemia to observe the physiological relevance of TXNIP modulation under metabolic disorders. RESULTS: TXNIP silencing did not mitigate the adverse effects on cell recruitment, vasculogenic properties, or senescence induced by metabolic stress in ECFCs. However, it significantly reduced IL-8 secretion and consequent neutrophil recruitment under these conditions. In a mouse model of hindlimb ischemia, endothelial deletion of TXNIP reduced MIP-2 secretion and prevented increased neutrophil recruitment induced by age-related comorbidities. CONCLUSIONS: Our findings suggest that targeting TXNIP in ECFCs may alleviate ischemic complications exacerbated by metabolic stress, offering potential clinical benefits for patients suffering from age-related comorbidities.
Subject(s)
Carrier Proteins , Interleukin-8 , Neutrophil Infiltration , Stress, Physiological , Animals , Interleukin-8/metabolism , Interleukin-8/genetics , Carrier Proteins/metabolism , Carrier Proteins/genetics , Humans , Mice , Neutrophil Infiltration/drug effects , Endothelial Progenitor Cells/metabolism , Endothelial Progenitor Cells/drug effects , Ischemia/metabolism , Ischemia/pathology , RNA, Small Interfering/metabolism , Thioredoxins/metabolism , Thioredoxins/genetics , Hindlimb/blood supply , Mice, Inbred C57BL , Glucose/metabolismABSTRACT
This study explores the impact of senescence on autocrine C-C motif chemokine ligand 5 (CCL5) in human endothelial progenitor cell (EPCs), addressing the poorly understood decline in number and function of EPCs during ageing. We examined the effects of replication-induced senescence on CCL5/CCL5 receptor (CCR5) signalling and angiogenic activity of EPCs in vitro and in vivo. We also explored microRNAs controlling CCL5 secretion in senescent EPCs, its impact on EPC angiogenic activity, and validated our findings in humans. CCL5 secretion and CCR5 levels in senescent EPCs were reduced, leading to attenuated angiogenic activity. CCL5 enhanced EPC proliferation via the CCR5/AKT/P70S6K axis and increased vascular endothelial growth factor (VEGF) secretion. Up-regulation of miR-409 in senescent EPCs resulted in decreased CCL5 secretion, inhibiting the angiogenic activity, though these negative effects were counteracted by the addition of CCL5 and VEGF. In a mouse hind limb ischemia model, CCL5 improved the angiogenic activity of senescent EPCs. Analysis involving 62 healthy donors revealed a negative association between CCL5 levels, age and Framingham Risk Score. These findings propose CCL5 as a potential biomarker for detection of EPC senescence and cardiovascular risk assessment, suggesting its therapeutic potential for age-related cardiovascular disorders.
Subject(s)
Cellular Senescence , Chemokine CCL5 , Endothelial Progenitor Cells , MicroRNAs , Neovascularization, Physiologic , Animals , Humans , Male , Mice , Angiogenesis , Cell Proliferation , Chemokine CCL5/metabolism , Chemokine CCL5/genetics , Down-Regulation/genetics , Endothelial Progenitor Cells/metabolism , Endothelial Progenitor Cells/cytology , Ischemia/metabolism , Ischemia/pathology , Ischemia/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Neovascularization, Physiologic/genetics , Receptors, CCR5/metabolism , Receptors, CCR5/genetics , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/geneticsABSTRACT
In the realm of large-area trauma flap transplantation, averting ischaemic necrosis emerges as a pivotal concern. Several key mechanisms, including the promotion of angiogenesis, the inhibition of oxidative stress, the suppression of cell death, and the mitigation of inflammation, are crucial for enhancing skin flap survival. Apoptotic bodies (ABs), arising from cell apoptosis, have recently emerged as significant contributors to these functions. This study engineered three-dimensional (3D)-ABs using tissue-like mouse adipose-derived stem cells (mADSCs) cultured in a 3D environment to compare their superior biological effects against 2D-ABs in bolstering skin flap survival. The findings reveal that 3D-ABs (85.74 ± 4.51) % outperform 2D-ABs (76.48 ± 5.04) % in enhancing the survival rate of ischaemic skin flaps (60.45 ± 8.95) % (all p < 0.05). Mechanistically, they stimulated angiogenesis, mitigated oxidative stress, suppressed apoptosis, and facilitated the transition of macrophages from M1 to M2 polarization (all p < 0.05). A comparative analysis of microRNA (miRNA) profiles in 3D- and 2D-ABs identified several specific miRNAs (miR-423-5p-up, miR30b-5p-down, etc.) with pertinent roles. In summary, ABs derived from mADSCs cultured in a 3D spheroid-like arrangement exhibit heightened biological activity compared to those from 2D-cultured mADSCs and are more effective in promoting ischaemic skin flap survival. These effects are attributed to their influence on specific miRNAs.
Subject(s)
Adipose Tissue , Apoptosis , Cell Culture Techniques , Ischemia , Stem Cells , Cells, Cultured , Humans , Animals , Mice , Stem Cells/cytology , Stem Cells/metabolism , Male , Mice, Inbred C57BL , Cell Culture Techniques/methods , Cell Separation/methods , Adipose Tissue/cytology , Adipose Tissue/metabolism , Ischemia/genetics , Ischemia/pathology , Cell Hypoxia , Cell Survival , MicroRNAs/genetics , Oxidative Stress , Neovascularization, Pathologic , Gene Expression ProfilingABSTRACT
Despite decades of effort aimed at developing clinically effective cell therapies, including mixed population mononuclear cells, to revascularize the ischemic limb, there remains a paucity of patient-based studies that inform the function and fate of candidate cell types. In this study, we showed that circulating proangiogenic/arteriogenic monocytes (PAMs) expressing the FcγIIIA receptor CD16 were elevated in patients with chronic limb-threatening ischemia (CLTI), and these amounts decreased after revascularization. Unlike CD16-negative monocytes, PAMs showed large vessel remodeling properties in vitro when cultured with endothelial cells and smooth muscle cells and promoted salvage of the ischemic limb in vivo in a mouse model of hindlimb ischemia. PAMs showed a propensity to migrate toward and bind to ischemic muscle and to secrete angiogenic/arteriogenic factors, vascular endothelial growth factor A (VEGF-A) and heparin-binding epidermal growth factor. We instigated a first-in-human single-arm cohort study in which autologous PAMs were injected into the ischemic limbs of five patients with CLTI. Greater than 25% of injected cells were retained in the leg for at least 72 hours, of which greater than 80% were viable, with evidence of enhanced large vessel remodeling in the injected muscle area. In summary, we identified up-regulation of a circulatory PAM subpopulation as an endogenous response to limb ischemia in CLTI and tested a potentially clinically relevant therapeutic strategy.
Subject(s)
Hindlimb , Ischemia , Monocytes , Neovascularization, Physiologic , Humans , Monocytes/metabolism , Animals , Ischemia/pathology , Ischemia/metabolism , Ischemia/therapy , Hindlimb/blood supply , Receptors, IgG/metabolism , Mice , Male , Vascular Endothelial Growth Factor A/metabolism , Female , Aged , Middle Aged , Cell Movement , Heparin-binding EGF-like Growth Factor/metabolismABSTRACT
Intestinal preservation for transplantation is accompanied by hypoperfusion with long periods of ischemia with total blood cessation and absolute withdrawal of oxygen leading to structural damage. The application of intraluminal oxygen has been successfully tested in small-animal series during storage and transport of the organ but have been so far clinically unrelatable. In this study, we tested whether a simple and clinically approachable method of intraluminal oxygen application could prevent ischemic damage in a large animal model, during warm ischemia time. We utilised a local no-flow ischemia model of the small intestine in pigs. A low-flow and high-pressure intraluminal oxygen deliverance system was applied in 6 pigs and 6 pigs served as a control group. Mucosal histopathology, hypoxia and barrier markers were evaluated after two hours of no-flow conditions, in both treatment and sham groups, and in healthy tissue. Macro- and microscopically, the luminal oxygen delivered treatment group showed preserved small bowel's appearance, viability, and mucosal integrity. A gradual deterioration of histopathology and barrier markers and increase in hypoxia-inducible factor 1-α expression towards the sites most distant from the oxygen application was observed. Intraluminal low-flow, high oxygen delivery can preserve the intestinal mucosa during total ischemia of the small intestine. This finding can be incorporated in methods to overcome small bowel ischemia and improve intestinal preservation for transplantation.
Subject(s)
Intestinal Mucosa , Intestine, Small , Ischemia , Oxygen , Animals , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestinal Mucosa/blood supply , Intestine, Small/metabolism , Intestine, Small/blood supply , Intestine, Small/pathology , Oxygen/metabolism , Swine , Ischemia/metabolism , Ischemia/pathology , Ischemia/therapy , Disease Models, Animal , Organ Preservation/methods , Hypoxia-Inducible Factor 1, alpha Subunit/metabolismABSTRACT
BACKGROUND: Current strategies for hypertrophic scar prevention and treatment are limited. OBJECTIVE: To facilitate these efforts, a minimally invasive hypertrophic scar model was created in a rabbit ear for the first time based on previous methods used to induce ischemia. METHODS: Six New Zealand white rabbits (12 ears total) were studied. First, ischemia was achieved by ligating the cranial artery, cranial vein and central artery, while preserving the caudal artery, caudal vein and central vein, respectively. The relative level of ischemia induced at time of surgery, both baseline and maximum perfusion, was assessed with a fluorescent light-assisted angiography and demonstrated lower rates of perfusion in the ischemic ears. Following vascular injury, a 2-cm full thickness linear wound was created on the ventral ear and closed with 4 - 0 Nylon sutures under high tension. For each rabbit, one ear received a combination of ischemia and wounding with suture tension (n = 6), while the other ear was non-ischemic with wounding and suture tension alone (n = 6). RESULTS: Four weeks post-operatively, ischemic ears developed scar hypertrophy (histological scar thickness: 1.1 ± 0.2 mm versus 0.5 ± 0.1 mm, p < 0.05). CONCLUSION: Herein, we describe a novel, prototypical minimally invasive rabbit ear model of hypertrophic scar formation that can allow investigation of new drugs for scar prevention.
Subject(s)
Cicatrix, Hypertrophic , Disease Models, Animal , Minimally Invasive Surgical Procedures , Animals , Rabbits , Cicatrix, Hypertrophic/pathology , Cicatrix, Hypertrophic/etiology , Cicatrix, Hypertrophic/prevention & control , Cicatrix, Hypertrophic/surgery , Minimally Invasive Surgical Procedures/methods , Minimally Invasive Surgical Procedures/adverse effects , Ear/surgery , Ear/pathology , Ischemia/etiology , Ischemia/surgery , Ischemia/pathology , Humans , Wound Healing , Suture TechniquesABSTRACT
BACKGROUND: Retinal ischemia plays a central pathophysiological role in numerous eye diseases, such as glaucoma. In addition to apoptosis, autophagy, necroptosis and ferroptosis are among the cell death mechanisms of ischemia; however, their role is not clearly understood and controversially discussed. OBJECTIVE: The aim of this study is to gain an improved understanding of the role of alternative cell death mechanisms such as autophagy and necroptosis after retinal ischemia. Based on this, future autophagy-based or necroptosis-based therapeutic approaches could be developed. MATERIAL AND METHODS: Retinal ischemia reperfusion was induced in one eye of 6 to 8week-old rats by temporarily increasing the intraocular pressure to 140â¯mmâ¯Hg (60â¯min), followed by reperfusion. The untreated contralateral eye served as a control. Retinas after ischemia and control retinas were examined 7 days after ischemia immunohistochemically with markers for retinal ganglion cells (RGC), astrocytes (GFAP) as well as an autophagy (LAMP1) and a necroptosis marker (RIPK3) (nâ¯= 6/group). RESULTS: Ischemia reperfusion resulted in both significant RGC loss (pâ¯≤ 0.001) and a significant increase of astrocyte area (pâ¯= 0.026) after 7 days. Interestingly, the number of autophagic LAMP1 positive cells was unchanged 7 days after ischemia (pâ¯= 0.272), whereas the number of necroptotic RIPK3 positive cells was significantly increased (pâ¯≤ 0.001). CONCLUSION: Necroptotic processes appear to be activated 7 days after ischemia reperfusion, contributing to retinal cell death and activation of astrocytes. Late autophagic processes are not activated 7 days after ischemia. Necroptosis-associated parameters could therefore be targeted as an early therapeutic approach after ischemia in the future.
Subject(s)
Necroptosis , Reperfusion Injury , Retinal Ganglion Cells , Animals , Rats , Retinal Ganglion Cells/pathology , Reperfusion Injury/pathology , Reperfusion Injury/metabolism , Autophagy/physiology , Male , Retinal Diseases/pathology , Retinal Diseases/metabolism , Retinal Diseases/physiopathology , Ischemia/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Apoptosis , Astrocytes/pathology , Astrocytes/metabolism , Glial Fibrillary Acidic Protein/metabolismABSTRACT
Avoiding ischemic necrosis after flap transplantation remains a significant clinical challenge. Developing an effective pretreatment method to promote flap survival postoperatively is crucial. Cobalt chloride (CoCl2) can increase cell tolerance to ischemia and hypoxia condition by stimulating hypoxia-inducible factor-1 (HIF-1) expression. However, the considerable toxic effects severely limit the clinical application of CoCl2. In this study, cobalt-based metal-organic frameworks (Co-MOF) encapsulated in a microneedle patch (Co-MOF@MN) was developed to facilitate the transdermal sustained release of Co2+ for rapid, minimally invasive rapid pretreatment of flap transplantation. The MN patch was composed of a fully methanol-based two-component cross-linked polymer formula, with a pyramid structure and high mechanical strength, which satisfied the purpose of penetrating the skin stratum corneum of rat back to achieve subcutaneous vascular area administration. Benefiting from the water-triggered disintegration of Co-MOF and the transdermal delivery via the MN patch, preoperative damage and side effects were effectively mitigated. Moreover, in both the oxygen-glucose deprivation/recovery (OGD/R) cell model and the rat dorsal perforator flap model, Co-MOF@MN activated the HIF-1α pathway and its associated downstream proteins, which reduced reperfusion oxidative damage, improved blood supply in choke areas, and increased flap survival rates post-transplantation. This preprotection strategy, combining MOF nanoparticles and the MN patch, meets the clinical demands for trauma minimization and uniform administration in flap transplantation. STATEMENT OF SIGNIFICANCE: Cobalt chloride (CoCl2) can stimulate the expression of hypoxia-inducible factor (HIF-1) and improve the tolerance of cells to ischemia and hypoxia conditions. However, the toxicity and narrow therapeutic window of CoCl2 severely limit its clinical application. Herein, we explored the role of Co-MOF as a biocompatible nanocage for sustained release of Co2+, showing the protective effect on vascular endothelial cells in the stress model of oxygen-glucose deprivation. To fit the clinical needs of minimal trauma in flap transplantation, a Co-MOF@MN system was developed to achieve local transdermal delivery at the choke area, significantly improving blood supply opening and flap survival rate. This strategy of two-step delivery of Co2+ realized the enhancement of biological functions while ensuring the biosafety.
Subject(s)
Cobalt , Metal-Organic Frameworks , Surgical Flaps , Animals , Humans , Male , Rats , Cobalt/chemistry , Cobalt/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ischemia/pathology , Metal-Organic Frameworks/chemistry , Metal-Organic Frameworks/pharmacology , Necrosis , Rats, Sprague-Dawley , Transdermal PatchABSTRACT
Critical limb ischemia (CLI) refers to a severe condition resulting from gradual obstruction in the supply of blood, oxygen, and nutrients to the limbs. The most promising clinical solution to CLI is therapeutic angiogenesis. This study explored the potency of pro-angiogenic terbium hydroxide nanorods (THNR) for treatment of CLI, with a major focus on their impact on ischemia-induced maladaptive alterations in endothelial cells as well as on vascularization in ischemic limbs. This study demonstrated that, in hypoxia-exposed endothelial cells, THNR improve survival and promote proliferation, migration, restoration of nitric oxide production, and regulation of vascular permeability. Based on molecular studies, these attributes of THNR can be traced to the stimulation of PI3K/AKT/eNOS signaling pathways. Besides, Wnt/GSK-3ß/ß-catenin signaling pathways may also play a role in the therapeutic actions of THNR. Furthermore, in the murine model of CLI, THNR administration can integrally re-establish blood perfusion with concomitant reduction of muscle damage and inflammation. Additionally, improvement of locomotor activities and motor coordination in ischemic limbs in THNR treated mice is also evident. Overall, the study demonstrates that THNR have the potential to be developed as an efficacious and cost-effective alternative clinical therapy for CLI, using a nanomedicine approach.
Subject(s)
Ischemia , Nanotubes , Animals , Nanotubes/chemistry , Mice , Ischemia/drug therapy , Ischemia/pathology , Ischemia/metabolism , Humans , Hydroxides/chemistry , Hydroxides/pharmacology , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Male , Materials Testing , Cell Proliferation/drug effects , Particle Size , Mice, Inbred C57BL , Human Umbilical Vein Endothelial Cells , Hindlimb/pathologyABSTRACT
Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit , Proteasome Endopeptidase Complex , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Animals , Humans , Mice , Proteasome Endopeptidase Complex/metabolism , Neovascularization, Physiologic , Proteolysis , Cell Hypoxia , Human Umbilical Vein Endothelial Cells/metabolism , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Male , Ischemia/metabolism , Ischemia/pathology , Angiogenesis , ProteinsABSTRACT
Type 2 diabetes mellitus (T2DM) significantly impairs the functionality and number of endothelial progenitor cells (EPCs) and resident endothelial cells, critical for vascular repair and regeneration, exacerbating the risk of vascular complications. GLP-1 receptor agonists, like dulaglutide, have emerged as promising therapeutic agents due to their multifaceted effects, including the enhancement of EPC activity and protection of endothelial cells. This study investigates dulaglutide's effects on peripheral blood levels of CD34+ and CD133+ cells in a mouse model of lower limb ischemia and its protective mechanisms against high-glucose-induced damage in endothelial cells. Results demonstrated that dulaglutide significantly improves blood flow, reduces tissue damage and inflammation in ischemic limbs, and enhances glycemic control. Furthermore, dulaglutide alleviated high-glucose-induced endothelial cell damage, evident from improved tube formation, reduced reactive oxygen species accumulation, and restored endothelial junction integrity. Mechanistically, dulaglutide mitigated mitochondrial fission in endothelial cells under high-glucose conditions, partly through maintaining SIRT1 expression, which is crucial for mitochondrial dynamics. This study reveals the potential of dulaglutide as a therapeutic option for vascular complications in T2DM patients, highlighting its role in improving endothelial function and mitochondrial integrity.
Subject(s)
Diabetes Mellitus, Experimental , Endothelial Progenitor Cells , Glucagon-Like Peptides , Glucose , Immunoglobulin Fc Fragments , Mitochondrial Dynamics , Recombinant Fusion Proteins , Sirtuin 1 , Animals , Mice , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Endothelial Progenitor Cells/drug effects , Endothelial Progenitor Cells/metabolism , Glucagon-Like Peptides/analogs & derivatives , Glucagon-Like Peptides/pharmacology , Glucagon-Like Peptides/therapeutic use , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Immunoglobulin Fc Fragments/pharmacology , Ischemia/metabolism , Ischemia/drug therapy , Ischemia/pathology , Mice, Inbred C57BL , Mitochondrial Dynamics/drug effects , Recombinant Fusion Proteins/pharmacology , Sirtuin 1/drug effects , Sirtuin 1/metabolismABSTRACT
Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.