Characterization of the Angiogenic Potential of Human Regulatory Macrophages (Mreg) after Ischemia/Reperfusion Injury In Vitro

Ischemia/reperfusion- (I/R-) induced organ damage represents one of the main causes of death worldwide, and new strategies to reduce I/R injury are urgently needed. We have shown that programmable cells of monocytic origin (PCMO) respond to I/R with the release of angiogenic mediators and that transplantation of PCMO results in increased neovascularization. Human regulatory macrophages (Mreg), which are also of monocytic origin, have been successfully employed in clinical transplantation studies due to their immunomodulatory properties. Here, we investigated whether Mreg also possess angiogenic potential in vitro and could represent a treatment option for I/R-associated illnesses. Mreg were differentiated using peripheral blood monocytes from different donors (N=14) by incubation with M-CSF and human AB serum and stimulation with INF-gamma. Mreg cultures were subjected to 3 h of hypoxia and 24 h of reoxygenation (resembling I/R) or the respective nonischemic control. Cellular resilience, expression of pluripotency markers, secretion of angiogenic proteins, and influence on endothelial tube formation as a surrogate marker for angiogenesis were investigated. Mreg showed resilience against I/R that did not lead to increased cell damage. Mreg express DHRS9 as well as IDO and display a moderate to low expression pattern of several pluripotency genes (e.g., NANOG, OCT-4, and SOX2). I/R resulted in an upregulation of IDO (p<0.001) while C-MYC and KLF4 were downregulated (p<0.001andp<0.05). Proteome profiling revealed the secretion of numerous angiogenic proteins by Mreg of which several were strongly upregulated by I/R (e.g., MIP-1alpha, 19.9-fold; GM-CSF, 19.2-fold; PTX3, 5.8-fold; IL-1β, 5.2-fold; and MCP-1, 4.7-fold). The angiogenic potential of supernatants from Mreg subjected to I/R remains inconclusive. While Mreg supernatants from 3 donors induced tube formation, 2 supernatants were not effective. We suggest that Mreg may prove beneficial as a cell therapy-based treatment option for I/R-associated illnesses. However, donor characteristics seem to crucially influence the effectiveness of Mreg treatment.

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Apigenin Protects the Brain against Ischemia/Reperfusion Injury via Caveolin-1/VEGF In Vitro and In Vivo

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/7017204 ◽

2018 ◽

Vol 2018 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Qiongyi Pang ◽

Yun Zhao ◽

Xiang Chen ◽

Kaiyi Zhao ◽

Qiongxiang Zhai ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Migration ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Tube Formation ◽

Caveolin 1 ◽

The Brain

Apigenin is a natural flavonoid found in several dietary plant foods as vegetables and fruits. To investigate potential anti-ischemia/reperfusion injury properties of apigenin in vitro, cell proliferation assay, tube formation, cell migration, apoptosis, and autophagy were performed in human brain microvascular endothelial cells (HBMVECs) after oxygen-glucose deprivation/reoxygenation (OGD/R). The effect of apigenin was also explored in rats after middle cerebral artery occlusion/reperfusion (MCAO/R) via neurobehavioral scores, pathological examination, and measurement of markers involved in ischemia/reperfusion injury. Data in vitro indicated that apigenin could prompt cell proliferation, tube formation, and cell migration while inhibiting apoptosis and autophagy by affecting Caveolin-1/VEGF, Bcl-2, Caspase-3, Beclin-1, and mTOR expression. Results in vivo showed that apigenin significantly reduced neurobehavioral scores and volume of cerebral infarction while prompting vascular endothelial cell proliferation by upregulating VEGFR2/CD34 double-labeling endothelial progenitor cell (EPC) number and affecting Caveolin-1, VEGF, and eNOS expression in brain tissue of MCAO/R rats. All the data suggested that apigenin may be protective for the brain against ischemia/reperfusion injury by alleviating apoptosis and autophagy, promoting cell proliferation in HBMVECs of OGD/R, and attenuating brain damage and improved neurological function in rats of MCAO/R through the Caveolin-1/VEGF pathway.

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Activation of the VEGF-A/ERK/PLA2 Axis Mediates Early Retinal Endothelial Cell Damage Induced by High Glucose: New Insight from an In Vitro Model of Diabetic Retinopathy

International Journal of Molecular Sciences ◽

10.3390/ijms21207528 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7528

Author(s):

Giovanni Giurdanella ◽

Gabriella Lupo ◽

Florinda Gennuso ◽

Federica Conti ◽

Debora Lo Furno ◽

...

Keyword(s):

High Glucose ◽

Cell Damage ◽

Tube Formation ◽

Blood Retinal Barrier ◽

Angiogenic Potential ◽

Normal Glucose ◽

Bromoenol Lactone ◽

Vitro Model ◽

Endothelial Growth Factor

Early blood retinal barrier (BRB) dysfunction induced by hyperglycemia was related to increased pro-inflammatory activity of phospholipase A2 (PLA2) and the upregulation of vascular endothelial growth factor A (VEGF-A). Here, we tested the role of VEGF-A in high glucose (HG)-induced damage of human retinal endothelial cells (HRECs) mediated by Ca++-dependent (cPLA2) and Ca++-independent (iPLA2) PLA2s. HRECs were treated with normal glucose (5 mM, NG) or high glucose (25 mM, HG) for 48 h with or without the VEGF-trap Aflibercept (Afl, 40 µg/mL), the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3; 15 µM), the iPLA2 inhibitor bromoenol lactone (BEL; 5 µM), or VEGF-A (80 ng/mL). Both Afl and AACOCF3 prevented HG-induced damage (MTT and LDH release), impairment of angiogenic potential (tube-formation), and expression of VEGF-A mRNA. Furthermore, Afl counteracted HG-induced increase of phospho-ERK and phospho-cPLA2 (immunoblot). VEGF-A in HG-medium increased glucose toxicity, through upregulation of phospho-ERK, phospho-cPLA2, and iPLA2 (about 55%, 45%, and 50%, respectively); immunocytochemistry confirmed the activation of these proteins. cPLA2 knockdown by siRNA entirely prevented cell damage induced by HG or by HG plus VEGF-A, while iPLA2 knockdown produced a milder protective effect. These data indicate that VEGF-A mediates the early glucose-induced damage in retinal endothelium through the involvement of ERK1/2/PLA2 axis activation.

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Epicatechin Gallate Protects HBMVECs from Ischemia/Reperfusion Injury through Ameliorating Apoptosis and Autophagy and Promoting Neovascularization

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/7824684 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Bing Fu ◽

Qinghong Zeng ◽

Zhaoting Zhang ◽

Mingyue Qian ◽

Jiechun Chen ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Migration ◽

Reperfusion Injury ◽

Green Tea ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Tube Formation ◽

Epicatechin Gallate

Green tea is one of the most beverages with antioxidants and nutrients. As one of the major components of green tea, (-)-epicatechin gallate (ECG) was evaluated for its antioxidative properties in the present study. Cell proliferation assay, tube formation, cell migration, apoptosis, and autophagy were performed in human brain microvascular endothelial cells (HBMVECs) after oxygen-glucose deprivation/reoxygenation (OGD/R) to investigate potential anti-ischemia/reperfusion injury properties of ECG in vitro. Markers of oxidative stress as ROS, LDH, MDA, and SOD were further assayed in our study. Data indicated that ECG could affect neovascularization and promote cell proliferation, tube formation, and cell migration while inhibiting apoptosis and autophagy through affecting VEGF, Bcl-2, BAX, LC3B, caspase 3, mTOR, and Beclin-1 expression. All the data suggested that ECG may be protective for the brain against ischemia/reperfusion injury by promoting neovascularization, alleviating apoptosis and autophagy, and promoting cell proliferation in HBMVECs of OGD/R.

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Gateways for Glutamate Neuroprotection in Parkinson’s Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD

Cells ◽

10.3390/cells9092037 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2037

Author(s):

Silvia Piccirillo ◽

Simona Magi ◽

Alessandra Preziuso ◽

Pasqualina Castaldo ◽

Salvatore Amoroso ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Ischemia Reperfusion Injury ◽

Excitatory Amino Acid ◽

Cell Damage ◽

Glutamate Uptake ◽

Ischemia Reperfusion ◽

Amino Acid Transporters ◽

Mitochondrial Functions

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson’s disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

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MiR-25 protects NRK-52E cells from apoptosis induced by renal IRI by targeting DKK3

10.21203/rs.3.rs-417907/v1 ◽

2021 ◽

Author(s):

Yu Zhang ◽

Xiangrong Zuo

Keyword(s):

Protective Effect ◽

Ischemia Reperfusion Injury ◽

Cell Damage ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Treatment Method ◽

Expression Level ◽

Iri Model ◽

Small Noncoding Rnas

Abstract Renal ischemia reperfusion injury (IRI) is one of the main causes of acute kidney injury (AKI). In recent years, there have been many studies on renal IRI, although an effective treatment method has not been developed. In recent years, growing evidence has shown that small noncoding RNAs play an important regulatory role in renal IRI. This article aims to explore whether microRNA-25 (miR-25) plays a role in the molecular mechanism of renal IRI. The results showed that the expression level of miR-25 was significantly downregulated in a rat renal IRI model, and this result was confirmed with in vitro experiments. After hypoxia-reoxygenation treatment, the apoptosis level of NRK-52E cells transfected with miR-25 mimics decreased significantly, and this antiapoptotic effect was antagonized by miR-25 inhibitors. In addition, we confirmed that DKK3 is a target of miR-25. MiR-25 exerts its protective effect against apoptosis on NRK-52E cells by inhibiting the expression of DKK3, and downregulating the expression level of miR-25 could disrupt this protective effect. In addition, we reconfirmed the role of miR-25 in rats. Therefore, we confirmed that miR-25 may target DKK3 to reduce renal cell damage caused by hypoxia and that miR-25 may be a new potential treatment for renal IRI.

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation

Cell Death and Disease ◽

10.1038/s41419-021-03622-x ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Ying Dong Du ◽

Wen Yuan Guo ◽

Cong Hui Han ◽

Ying Wang ◽

Xiao Song Chen ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Mitochondrial Fragmentation ◽

Hepatic Ischemia ◽

Adeno Associated Virus ◽

Downstream Target ◽

Hepatic Ischemia Reperfusion

AbstractDespite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.

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The Sodium-Glucose Cotransporter-2 Inhibitor Canagliflozin Alleviates Endothelial Dysfunction Following In Vitro Vascular Ischemia/Reperfusion Injury in Rats

International Journal of Molecular Sciences ◽

10.3390/ijms22157774 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7774

Author(s):

Sevil Korkmaz-Icöz ◽

Cenk Kocer ◽

Alex A. Sayour ◽

Patricia Kraft ◽

Mona I. Benker ◽

...

Keyword(s):

Endothelial Dysfunction ◽

Reperfusion Injury ◽

Vascular Function ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Artery Bypass ◽

Diabetic Rats ◽

Organ Bath ◽

Sodium Glucose Cotransporter

Vascular ischemia/reperfusion injury (IRI) contributes to graft failure and adverse clinical outcomes following coronary artery bypass grafting. Sodium-glucose-cotransporter (SGLT)-2-inhibitors have been shown to protect against myocardial IRI, irrespective of diabetes. We hypothesized that adding canagliflozin (CANA) (an SGLT-2-inhibitor) to saline protects vascular grafts from IRI. Aortic rings from non-diabetic rats were isolated and immediately mounted in organ bath chambers (control, n = 9–10 rats) or underwent cold ischemic preservation in saline, supplemented either with a DMSO vehicle (IR, n = 8–10 rats) or 50µM CANA (IR + CANA, n = 9–11 rats). Vascular function was measured, the expression of 88 genes using PCR-array was analyzed, and feature selection using machine learning was applied. Impaired maximal vasorelaxation to acetylcholine in the IR-group compared to controls was significantly ameliorated by CANA (IR 31.7 ± 3.2% vs. IR + CANA 51.9 ± 2.5%, p < 0.05). IR altered the expression of 17 genes. Ccl2, Ccl3, Ccl4, CxCr4, Fos, Icam1, Il10, Il1a and Il1b have been found to have the highest interaction. Compared to controls, IR significantly upregulated the mRNA expressions of Il1a and Il6, which were reduced by 1.5- and 1.75-fold with CANA, respectively. CANA significantly prevented the upregulation of Cd40, downregulated NoxO1 gene expression, decreased ICAM-1 and nitrotyrosine, and increased PECAM-1 immunoreactivity. CANA alleviates endothelial dysfunction following IRI.

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Overexpression of SP1 restores autophagy to alleviate acute renal injury induced by ischemia-reperfusion through the miR-205/PTEN/Akt pathway

Journal of Inflammation ◽

10.1186/s12950-021-00270-y ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Chong Huang ◽

Yan Chen ◽

Bin Lai ◽

Yan-Xia Chen ◽

Cheng-Yun Xu ◽

...

Keyword(s):

Reperfusion Injury ◽

Cell Apoptosis ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Renal Ischemia ◽

Akt Pathway ◽

Acute Renal Injury ◽

Injury Model ◽

Renal Ischemia Reperfusion Injury

Abstract Background Acute kidney injury (AKI) is a major kidney disease with poor clinical outcome. SP1, a well-known transcription factor, plays a critical role in AKI and subsequent kidney repair through the regulation of various cell biologic processes. However, the underlying mechanism of SP1 in these pathological processes remain largely unknown. Methods An in vitro HK-2 cells with anoxia-reoxygenation injury model (In vitro simulated ischemic injury disease) and an in vivo rat renal ischemia-reperfusion injury model were used in this study. The expression levels of SP1, miR-205 and PTEN were detected by RT-qPCR, and the protein expression levels of SP1, p62, PTEN, AKT, p-AKT, LC3II, LC3I and Beclin-1 were assayed by western blot. Cell proliferation was assessed by MTT assay, and the cell apoptosis was detected by flow cytometry. The secretions of IL-6 and TNF-α were detected by ELISA. The targeted relationship between miR-205 and PTEN was confirmed by dual luciferase report assay. The expression and positioning of LC-3 were observed by immunofluorescence staining. TUNEL staining was used to detect cell apoptosis and immunohistochemical analysis was used to evaluate the expression of SP1 in renal tissue after ischemia-reperfusion injury in rats. Results The expression of PTEN was upregulated while SP1 and miR-205 were downregulated in renal ischemia-reperfusion injury. Overexpression of SP1 protected renal tubule cell against injury induced by ischemia-reperfusion via miR-205/PTEN/Akt pathway mediated autophagy. Overexpression of SP1 attenuated renal ischemia-reperfusion injury in rats. Conclusions SP1 overexpression restored autophagy to alleviate acute renal injury induced by ischemia-reperfusion through the miR-205/PTEN/Akt pathway.

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Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

Molecular Medicine ◽

10.1186/s10020-021-00332-0 ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Jian-Ping Zhang ◽

Wei-Jing Zhang ◽

Miao Yang ◽

Hua Fang

Keyword(s):

Cell Apoptosis ◽

Ischemia Reperfusion Injury ◽

Glycogen Synthase ◽

Ischemia Reperfusion ◽

Inflammatory Factors ◽

Therapeutic Effects ◽

Protective Effects ◽

Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

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