Fabrication of Vascular Grafts Using Poly(ε-Caprolactone) and Collagen-Encapsuled ADSCs for Interposition Implantation of Abdominal Aorta in Rhesus Monkeys.

The production of small-diameter artificial vascular grafts continues to encounter numerous challenges, with concerns regarding the degradation rate and endothelialization being particularly critical. In this study, porous PCL scaffolds were prepared, and PCL vascular grafts were fabricated by 3D bioprinting of collagen materials containing adipose-derived mesenchymal stem cells (ADSCs) on the internal wall of the porous PCL scaffold. The PCL vascular grafts were then implanted in the abdominal aorta of Rhesus monkeys for up to 640 days to analyze the degradation of the scaffolds and regeneration of the aorta. Changes in surface morphology, mechanical properties, crystallization property, and molecular weight of porous PCL revealed a similar degradation process of PCL in PBS at pH 7.4 containing Thermomyces lanuginosus lipase and in situ in the abdominal aorta of rhesus monkeys. The contrast of in vitro and in vivo degradation provided valuable reference data for predicting in vivo degradation based on in vitro enzymatic degradation of PCL for further optimization of PCL vascular graft fabrication. Histological analysis through hematoxylin and eosin (HE) staining and fluorescence immunostaining demonstrated that the PCL vascular grafts successfully induced vascular regeneration in the abdominal aorta over the 640-day period. These findings provided valuable insights into the regeneration processes of the implanted vascular grafts. Overall, this study highlights the significant potential of PCL vascular grafts for the regeneration of small-diameter blood vessels.

Implantation of Adipose-Derived Mesenchymal Stromal Cells (ADSCs)-Lining Prosthetic Graft Promotes Vascular Regeneration in Monkeys and Pigs.

BACKGROUND: Current replacement procedures for stenosis or occluded arteries using prosthetic grafts have serious limitations in clinical applications, particularly, endothelialization of the luminal surface is a long-standing unresolved problem. METHOD: We produced a cell-based hybrid vascular graft using a bioink engulfing adipose-derived mesenchymal stromal cells (ADSCs) and a 3D bioprinting process lining the ADSCs on the luminal surface of GORE-Tex grafts. The hybrid graft was implanted as an interposition conduit to replace a 3-cm-long segment of the infrarenal abdominal aorta in Rhesus monkeys. RESULTS: Complete endothelium layer and smooth muscle layer were fully developed within 21 days post-implantation, along with normalized collagen deposition and crosslinking in the regenerated vasculature in all monkeys. The regenerated blood vessels showed normal functionality for the longest observation of more than 1650 days. The same procedure was also conducted in miniature pigs for the interposition replacement of a 10-cm-long right iliac artery and showed the same long-term effective and safe outcome. CONCLUSION: This cell-based vascular graft is ready to undergo clinical trials for human patients.

Alteration of endothelial permeability ensures cardiomyocyte survival from ischemic insult in the subendocardium of the heart.

Previous studies have shown that cardiomyocytes in the subendocardial region of myocardium survive from ischemic insult. This study was undertaken to explore possible mechanisms for the survival of these cardiomyocytes, focusing on changes in endothelial cells (ECs) and blood supply. C57/B6 mice were subjected to permanent ligation of left anterior descending (LAD) coronary artery to induce myocardial ischemia (MI). The hearts were harvested at 1, 4, and 7 days post MI and examined for histological changes. It was found that the survival of cardiomyocytes was associated with a preservation of ECs in the subendocardial region, as revealed by EC-specific tdTomato expression transgenic mice (Tie2tdTomato). However, the EC selective proteins, PECAM1 and VEGFR2, were significantly depressed in these ECs. Consequently, the ratio of PECAM1/tdTomato was significantly decreased, indicating a transformation from PECAM1+ ECs to PECAM1- ECs. Furthermore, EC junction protein, VE-cadherin, was not only depressed but also disassociated from PECAM1 in the same region. These changes led to an increase in EC permeability, as evidenced by increased blood infiltration in the subendocardial region. Thus, the increase in the permeability of ECs due to their transformation in the subendocardial region allows blood infiltration, creating a unique microenvironment and ensuring the survival of cardiomyocytes under ischemic conditions.

ABCE1 selectively promotes HIF-1α transactivation of angiogenic gene expression.

BACKGROUND: Copper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1). OBJECTIVE: The present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1α accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1α and FIH-1 was validated by Co-Immunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1α transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR. RESULTS: ATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu-binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2. CONCLUSION: The results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1α, thus promoting HIF-1 transactivation of angiogenic gene expression.

Protocol to assess fatal embolism risks from human stem cells.

Here, we present a protocol to identify the pro-embolic sub-population of human adipose-derived multipotent stromal cells (ADSCs) and predict fatal embolism risks from ADSC infusion. We describe steps for the collection, processing, and classification of ADSC single-cell RNA-seq data. We then detail the development of a mathematical model for predicting ADSC embolic risk. This protocol allows for the development of prediction models to enhance the assessment of cell quality and advance the clinical applications of stem cells. For complete details on the use and execution of this protocol, please refer to Yan et al. (2022).1.

Zinc-glutathione in Chinese Baijiu prevents alcohol-associated liver injury.

Zinc depletion is associated with alcohol-associated liver injury. We tested the hypothesis that increasing zinc availability along with alcohol consumption prevents alcohol-associated liver injury. Zinc-glutathione (ZnGSH) was synthesized and directly added to Chinese Baijiu. Mice were administered a single gastric dose of 6 g/kg ethanol in Chinese Baijiu with or without ZnGSH. ZnGSH in Chinese Baijiu did not change the likeness of the drinkers but significantly reduced the recovery time from drunkenness along with elimination of high-dose mortality. ZnGSH in Chinese Baijiu decreased serum AST and ALT, suppressed steatosis and necrosis, and increased zinc and GSH concentrations in the liver. It also increased alcohol dehydrogenase and aldehyde dehydrogenase in the liver, stomach, and intestine and reduced acetaldehyde in the liver. Thus, ZnGSH in Chinese Baijiu prevents alcohol-associated liver injury by increasing alcohol metabolism timely with alcohol consumption, providing an alternative approach to the management of alcohol-associated drinking.

Gene expression patterns and related pathways in the hearts of rhesus monkeys subjected to prolonged myocardial ischemia.

After myocardial infarction (MI) occurs, progressive pathological cardiac remodeling results in heart dysfunction and even heart failure during the following months or years. The present study explored the molecular mechanisms underlying the late phase of MI at the global transcript level. A rhesus monkey model of myocardial ischemia induced by left anterior descending (LAD) artery ligation was established, and the heart tissue was collected eight weeks after ligation for transcriptome analysis by DNA microarray technology. Differentially expressed genes in the core infarcted area and remote infarcted area of the ischemic heart were detected with significance analysis of microarray (SAM), and related pathways were detected by Gene Ontology (GO)/pathway analysis. We found that compared to the sham condition, prolonged ischemia increased the levels of 941 transcripts, decreased the levels of 380 transcripts in the core infarcted area, and decreased the levels of 8 transcripts in the remote area in monkey heart tissue. Loss of coordination between the expression of genes, including natriuretic peptide A (NPPA), NPPB, and corin (Corin, serine peptidase), may aggravate cardiac remodeling. Furthermore, imbalance in the enriched significantly changed pathways, including fibrosis-related pathways, cardioprotective pathways, and the cardiac systolic pathway, likely also plays a key role in regulating the development of heart remodeling.

Dose-specific efficacy of adipose-derived mesenchymal stem cells in septic mice.

BACKGROUND: Mesenchymal stem cells (MSCs) therapy for sepsis has been extensively studied in the past decade; however, the treatment regimen and mechanism of action of MSCs remain elusive. Here, we attempted to understand the efficacy and mechanism of action of MSCs on rescuing mice with sepsis. METHODS: A mouse model of sepsis was produced by cecal ligation and puncture (CLP). Allogeneic adipose-derived MSCs (ADSCs) were administered by intravenous infusion at 6 h after CLP, and dose-related effects of ADSCs on these mice were determined by survival rate, histopathological changes, biochemical and coagulation parameters, bacterial load, and plasma levels of endotoxin and inflammatory cytokines. The tissue distribution of intravenously infused ADSCs in septic mice was investigated by pre-labeling ADSCs with the lipophilic membrane dye PKH26. RNA sequencing analysis was performed to assess the transcriptional changes in peripheral blood mononuclear cells (PBMCs) and the liver. RESULTS: A significant therapeutic effect of ADSCs at a dose of 2 × 107 cells/kg in septic mice was evidenced by a remarkable reduction in mortality (35.89% vs. 8.89% survival rate), blood bacterial burden, systemic inflammation, and multiple organ damage. In contrast, ADSCs at a lower dose (1 × 107 cells/kg) failed to achieve any beneficial outcomes, while ADSCs at a higher dose (4 × 107 cells/kg) caused more early death within 24 h after CLP, retaining a steady survival rate of 21.42% thereafter. PKH26-labeled ADSCs were predominantly localized in the lungs of septic mice after intravenous infusion, with only a smaller proportion of PKH26-positive signals appearing in the liver and spleen. RNA sequencing analysis identified that insufficient phagocytic activity of PBMCs in addition to a hyperactivation of the hepatic immune response was responsible for the ineffectiveness of low-dose ADSCs therapy, and acute death caused by high-dose ADSCs infusion was associated with impaired coagulation signaling in PBMCs and exacerbated hepatic hypoxic injury. CONCLUSIONS: Our findings demonstrate a dose-specific effect of ADSCs on the treatment of sepsis due to dose-related interactions between exogenous stem cells and the host's microenvironment. Therefore, a precise dosing regimen is a prerequisite for ADSCs therapy for sepsis.

A clinical feasible stem cell encapsulation ensures an improved wound healing.

Cell encapsulation has proven to be promising in stem cell therapy. However, there are issues needed to be addressed, including unsatisfied yield, unmet clinically friendly formulation, and unacceptable viability of stem cells after cryopreservation and thawing. We developed a novel biosynsphere technology to encapsulate stem cells in clinically-ready biomaterials with controlled microsphere size. We demonstrated that biosynspheres ensure the bioviability and functionality of adipose-derived stromal cells (ADSCs) encapsulated, as delineated by a series of testing procedures. We further demonstrated that biosynspheres protect ADSCs from the hardness of clinically handling such as cryopreservation, thawing, high-speed centrifugation and syringe/nozzle injection. In a swine full skin defect model, we showed that biosynspheres were integrated to the destined tissues and promoted the repair of injured tissues with an accelerating healing process, less scar tissue formation and normalized deposition of collagen type I and type III, the ratio similar to that found in normal skin. These findings underscore the potential of biosynsphere as an improved biofabrication technology for tissue regeneration in clinical setting.

Alteration of Transcriptomic Profile and Antiseptic Efficacy of Adipose-Derived Mesenchymal Stromal/Stem Cells Under Different Culture Conditions.

Mesenchymal stromal/stem cells (MSCs) are a promising therapeutic agent for various diseases, including sepsis. However, translating MSC therapy to clinical applications remains challenging due to variations in the properties of MSCs under different preparation conditions. In this study, the gene expression profiles of human adipose-derived mesenchymal stromal/stem cells (ADSCs) under different culture conditions were compared in relation to their therapeutic efficacy for sepsis. Results showed that ADSCs cultured in media supplemented with human platelet lysates (hPL) (hPL-ADSCs) exhibited a smaller cell size and higher proliferative capacity, whereas ADSCs cultured in media supplemented with fetal bovine serum (FBS) (FBS-ADSCs) showed a broader and flatter shape. Both hPL-ADSCs and FBS-ADSCs exhibited a protective effect in a mouse model of sepsis; however, hPL-ADSCs displayed a better potency for immunosuppressive function, as evidenced by a better improvement of survival rate and further reduction of tissue injury and infectious biomarkers (alanine transaminase and procalcitonin). Furthermore, hPL-ADSCs caused a more anti-inflammatory transcriptomic shift, whereas FBS-ADSCs led to more depression of proinflammatory transcriptomic response. This study thus demonstrates that both hPL-ADSCs and FBS-ADSCs are effective for antiseptic therapy via different mechanisms of inflammatory manipulation, although hPL-ADSCs may imply a better preference.

Transcriptomic heterogeneity of cultured ADSCs corresponds to embolic risk in the host.

Stem cell therapy emerges as an effective approach for treating various currently untreatable diseases. However, fatal and unknown risks caused by their systemic use remain to be a major obstacle to clinical application. We developed a functional single-cell RNA sequencing (scRNA-seq) procedure and identified that transcriptomic heterogeneity of adipose-derived stromal cells (ADSCs) in cultures is responsible for a fatal embolic risk of these cells in the host. The pro-embolic subpopulation of ADSCs in cultures was sorted by gene set enrichment analysis (GSEA) and verified by a supervised machine learning analysis. A mathematical model was developed and validated for the prediction of embolic risk of cultured ADSCs in animal models and further confirmed by its application to public data. Importantly, modification of culture conditions prevented the embolic risk. This novel procedure can be applied to other aspects of risk assessment and would help further the development of stem cell clinical applications.

An Improved Monkey Model of Myocardial Ischemic Infarction for Cardiovascular Drug Development.

Non-human primate monkey model of myocardial ischemic infarction is precious for translational medicine research. Ligation of the left anterior descending (LAD) artery is a common procedure to induce myocardial ischemic infarction. However, the consistency of the myocardial infarction thus generated remains problematic. The present study was undertaken to critically evaluate the monkey model of myocardial ischemic infarction to develop a procedure for a consistent cross-study comparison. Forty male Rhesus monkeys were divided into 4 groups and subjected to LAD artery ligation at different levels along the artery. In addition, the major diagonal branch was selectively ligated parallel to the ligation site of the LAD artery according to the diagonal branch distribution. Analyses of MRI, echocardiography, cardiac hemodynamics, electrocardiography, histopathology, and cardiac injury biomarkers were undertaken to characterize the monkeys with myocardial infarction. Ligation at 40% of the total length of the artery, measured from the apex end, produced variable infarct areas with inconsistent functional alterations. Ligation at 60% or above coupled with selective ligation of diagonal branches produced a consistent myocardial infarction with uniform dysfunction. However, ligation at 70% caused a lethal threat. After a thorough analysis, it is concluded that ligation at 60% of the total length coupled with selective ligation of diagonal branches, enables standardization of the location of occlusion and the subsequent ischemic area, as well as avoids the influence of the diagonal branches, are ideal to produce a consistent monkey model of myocardial ischemic infarction.

A novel method for histological examination of hair follicles.

Intact and healthy hair follicles are important for hair growth after hair follicle transplantation. However, effective and practical evaluation methods for the quality of hair follicles are currently lacking. In the present study, we developed a novel fast staining method for histological examination of hair follicles. The whisker follicles from mice were used to explore the staining protocols, and the final protocol for the evaluation of human hair follicles was derived from animal experiments. After extraction, human hair follicles or mouse whisker follicles were permeabilized with 0.3% Triton X-100. Subsequently, hair follicles were processed by either hematoxylin or alkaline phosphatase staining. The integrity and growth state, including the status of hair follicle stem cells and blood vessels of the extracted hair follicles, were clearly identified under a light microscope. Unhealthy hair follicles from donors or hair follicles broken during extraction were easily revealed by this method. Importantly, it took less than half an hour to obtain images of an individual hair follicle. This method is simple and practical for evaluating the quality and status of hair follicles, providing a fast-screening procedure for hair follicle transplantation.

Atherosclerotic lesion-specific copper delivery suppresses atherosclerosis in high-cholesterol-fed rabbits.

Dietary cholesterol supplements cause hypercholesterolemia and atherosclerosis along with a reduction of copper concentrations in the atherosclerotic wall in animal models. This study was to determine if target-specific copper delivery to the copper-deficient atherosclerotic wall can block the pathogenesis of atherosclerosis. Male New Zealand white rabbits, 10-weeks-old and averaged 2.0 kg, were fed a diet containing 1% (w/w) cholesterol or the same diet without cholesterol as control. Twelve weeks after the feeding, the animals were injected with copper-albumin microbubbles and subjected to ultrasound sonication specifically directed at the atherosclerotic lesions (Cu-MB-US) for target-specific copper delivery, twice a week for four weeks. This regiment was repeated 3 times with a gap of two weeks in between. Two weeks after the last treatment, the animals were harvested for analyses of serum and aortic pathological changes. Compared to controls, rabbits fed cholesterol-rich diet developed atherosclerotic lesion with a reduction in copper concentrations in the lesion tissue. Cu-MB-US treatment significantly increased copper concentrations in the lesion, and reduced the size of the lesion. Furthermore, copper repletion reduced the number of apoptotic cells as well as the content of cholesterol and phospholipids in the atherosclerotic lesion without a disturbance of the stability of the lesion. The results thus demonstrate that target-specific copper supplementation suppresses the progression of atherosclerosis at least in part through preventing endothelial cell death, thus reducing lipid infiltration in the atherosclerotic lesion.

Extracellular matrix remodeling is associated with the survival of cardiomyocytes in the subendocardial region of the ischemic myocardium.

A significant amount of cardiomyocytes in subendocardial region survive from ischemic insults. In order to understand the mechanism by which these cardiomyocytes survive, the present study was undertaken to examine changes in these surviving cardiomyocytes and their extracellular matrix. Male C57BL/6 mice aged 8-12 weeks old were subjected to a permanent left anterior descending coronary artery ligation to induce ischemic injury. The hearts were collected at 1, 4, 7, or 28 days after the surgery and examined by histology. At day 1 after left anterior descending ligation, there was a significant loss of cardiomyocytes through apoptosis, but a proportion of cardiomyocytes were surviving in the subendocardial region. The surviving cardiomyocytes were gradually changed from rod-shaped to round-shaped, and appeared disconnected. Connexin 43, an important gap junction protein, was significantly decreased, and collagen I and III deposition was significantly increased in the extracellular matrix. Furthermore, lysyl oxidase, a copper-dependent amine oxidase catalyzing the cross-linking of collagens, was significantly increased in the extracellular matrix, paralleled with the surviving cardiomyocytes. Inhibition of lysyl oxidase activity reduced the number of surviving cardiomyocytes. Thus, the extracellular matrix remodeling is correlated with the deformation of cardiomyocytes, and the electrical disconnection between the surviving cardiomyocytes due to connexin 43 depletion and the increase in lysyl oxidase would help these deformed cardiomyocytes survive under ischemic conditions.

Selective suppression of M1 macrophages is involved in zinc inhibition of liver fibrosis in mice.

Zinc deficiency is common in the liver of patients with chronic liver disease. Zinc supplementation suppresses the progression of liver fibrosis induced by bile duct ligation (BDL) in mice. The present study was undertaken to specifically investigate a possible mechanism by which zinc plays this role in the liver. Kunming mice were subjected to BDL for 4 weeks to induce liver fibrosis, and concomitantly treated with zinc sulfite or saline as control by gavage once a day. The results showed that zinc supplementation significantly suppressed liver fibrosis and inflammation along with inhibition of hepatic stellate cells activation induced by BDL. These inhibitory effects were accompanied by the reduction of collagen deposition and a significant reduction of macrophage infiltration affected livers. Importantly, zinc selectively inhibited M1 macrophage polarization and M1-related inflammatory cytokines. This inhibitory effect was further confirmed by the reduction of relevant biomarkers of M1 macrophages including inducible NO synthase (iNOS), monocyte chemotactic protein-1 (MCP-1/CCL2), and tumor necrosis factor-α in the zinc supplemented BDL livers. In addition, zinc inhibition of M1 macrophages was associated with a decrease of Notch1 expression. Taken together, these data indicated that zinc supplementation inhibited liver inflammation and fibrosis in BDL mice through selective suppression of M1 macrophages, which is associated with inhibition of Notch1 pathway in M1 macrophage polarization.

FLI1 mediates the selective expression of hypoxia-inducible factor 1 target genes in endothelial cells under hypoxic conditions.

The selective expression of hypoxia-inducible factor (HIF) target genes in different physiological and pathological environments forms the basis for cellular adaptation to hypoxia in development and disease. Several E26 transformation-specific (ETS) transcription factors have been shown to specifically regulate the expression of a subset of HIF-2 target genes. However, it is unknown whether there are ETS factors that specifically regulate hypoxia-induced HIF-1 target genes. The present study was undertaken to explore whether friend leukemia integration 1 (FLI1), an ETS transcription factor, regulates the expression of HIF-1 target genes. To investigate this possibility, EA.hy926 cells were exposed to 20% O2 (normoxia) or 1% O2 (hypoxia). Western blotting, immunofluorescence staining, and RT-qPCR revealed that FLI1 mRNA and protein levels increased slightly and that the FLI1 protein co-localized with HIF-1α in the nucleus under hypoxic conditions. Further analysis showed that, in the absence of FLI1, the hypoxia-mediated induction of HIF-1 target genes was selectively inhibited. The results from immunoprecipitation and luciferase reporter assays indicated that FLI1 cooperates with HIF-1α and is required for the transcriptional activation of a subset of HIF-1 target genes with a core promoter region containing FBS in proximity to a functional hypoxia response element (HRE). Furthermore, ChIP analysis further confirmed the direct interaction between FLI1 and the promoter region of FLI1-dependent HIF-1 target genes under hypoxia. Together, this study demonstrates that FLI1 is involved in the transactivation of certain HIF-1 target genes in endothelial cells under hypoxic conditions.

Copper Preserves Vasculature Structure and Function by Protecting Endothelial Cells from Apoptosis in Ischemic Myocardium.

The present study was undertaken to investigate whether Cu protects vasculatures from ischemic injury in the heart. C57/B6 mice were introduced to myocardial ischemia (MI) by permanent ligation of the left anterior descending (LAD) coronary artery. Two hours post-LAD ligation, mice were intravenously injected with a Cu-albumin (Cu-alb) solution, or saline as control. At 1, 4, or 7 days post-MI, hearts were collected for further analysis. A dramatic decrease in CD31-positive endothelial cells concomitantly with abundant apoptosis, along with obstruction of blood flow, was observed in ischemic myocardium 1 day post-MI. The early Cu-alb treatment protected CD31-positive cells from apoptosis, along with a preservation of micro-vessels and a decrease in infarct size. This early vasculature preservation ensured myocardial blood perfusion and protected cardiac contractile function until 28 days post-MI. This strategy of Cu-alb treatment immediately following MI would help develop a therapeutic approach for acute heart attack patients in a clinical setting.

Cardiomyocyte-Specific COMMD1 Deletion Suppresses Ischemia-Induced Myocardial Apoptosis.

Copper metabolism MURR domain 1 (COMMD1) increases in ischemic myocardium along with suppressed contractility. Cardiomyocyte-specific deletion of COMMD1 preserved myocardial contractile function in response to the same ischemic insult. This study was undertaken to test the hypothesis that cardiomyocyte protection in COMMD1 myocardium is responsible for the functional preservation of the heart in response to ischemic insult. After ischemic insult, there were significantly more cardiomyocytes in the cardiomyocyte-specific COMMD1 deletion myocardium than that in WT controls. This preservation of cardiomyocytes was paralleled by a significant suppression of apoptosis in the COMMD1 deletion myocardium compared to controls. In searching for the mechanistic understanding of the anti-apoptotic effect of COMMD1 deletion, we found the anti-apoptotic Bcl-2 mRNA and protein expression were upregulated and the pro-apoptotic Bax mRNA and protein expression were downregulated. The critical transcription factor RelA, maintaining a high ratio between Bcl-2 and Bax for anti-apoptotic action, was suppressed by ischemia, but was rescued in the COMMD1 deletion myocardium. Because COMMD1 is critically involved in RelA ubiquitination and degradation, the data obtained here demonstrate that COMMD1 deletion leads to RelA preservation in ischemic myocardium, promoting the Bcl-2 anti-apoptotic pathway and suppressing the Bax pro-apoptotic pathway, and in combination, leading to protection of cardiomyocytes from ischemia-induced apoptosis.

The Association of Suppressed Hypoxia-Inducible Factor-1 Transactivation of Angiogenesis With Defective Recovery From Cerebral Ischemic Injury in Aged Rats.

Elderly patients suffer more brain damage in comparison with young patients from the same ischemic stroke. The present study was undertaken to test the hypothesis that suppressed hypoxia-inducible factor-1 (HIF-1) transcription activity is responsible for defective recovery after ischemic stroke in the elders. Aged and young rats underwent 1-h transient middle cerebral artery occlusion (MCAO) to produce cerebral ischemic injury. The initial cerebral infarct volume in the young gradually declined as time elapsed, but in the aged rats remained the same. The defective recovery in the aged was associated with depressed angiogenesis and retarded neurorestoration. There was no difference in HIF-1α accumulation in the brain between the two age groups, but the expression of HIF-1 regulated genes involved in cerebral recovery was suppressed in the aged. In confirmation, inhibition of HIF-1 transactivation of gene expression in the young suppressed cerebral recovery from MCAO as the same as that observed in the aged rats. Furthermore, a copper metabolism MURR domain 1 (COMMD1) was significantly elevated after MCAO only in the brain of aged rats, and suppression of COMMD1 by siRNA targeting COMMD1 restored HIF-1 transactivation and improved recovery from MCAO-induced damage in the aged brain. These results demonstrate that impaired HIF-1 transcription activity, due at least partially to overexpression of COMMD1, is associated with the defective cerebral recovery from ischemic stroke in the aged rats.