Infarct Area Research Articles

Cardioprotective Effect of Nano Gold Kalimag Injection via Inhibiting Oxidative Stress and Improving Myocardial Energy Metabolism in Ischemic Heart Injury Model

Background: Many people worldwide die from cardiovascular diseases. Myocardial ischemia can be induced by atherosclerosis, and the main factor responsible for atherosclerosis is the deposition of lipid peroxide in vessels. Therefore, atherogenesis may be prevented and treated at the cellular level by preventing oxidative stress using the properties of nanogold, magnesium and potassium. In this study, we have elucidated the cardioprotective effect of Nano Gold Kalimag injection (NGKM) in the ischemic heart injury model in rats. Methods: The ischemic heart injury models were made with adrenaline and pituitrin, and a few myocardial damage-related parameters of the models were observed after NGKM injection. After NGKM injection, the levels of lactate dehydrogenase (LDH), aspartate transamiase (AST), and creatine kinase (CK) in an ischemic heart injury model in rats were detected, and TTC staining and H&E staining were performed. In addition, in the myocardial tissues of rats, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase, and Na+-K+-ATPase activity were measured, and lactate content and ATP content were measured. Results: NGKM can reduce the infarct area in the ischemic heart injury model and significantly lower LDH, AST, and CK levels. SOD, GSH-Px, and arterial tissue CAT levels significantly increased compared to the model, and MDA contents significantly decreased in myocardial tissue compared to the model group. In addition, myocardial tissue ATP content and Na+-K+-ATPase level in myocardial tissues significantly increased compared to the model group, and lactate content in myocardial tissue significantly decreased compared to the model group. Conclusions: Our findings suggest that NGKM injection has cardioprotective effects by inhibiting oxidative stress in an ischemic heart injury model in rats.

A cell-penetrating peptide exerts therapeutic effects against ischemic stroke by mediating the lysosomal degradation of sirtuin 5.

Stroke is a major public health concern worldwide. The lack of effective therapies heightens the need for new therapeutic agents. Previous study identified sirtuin 5 (SIRT5) as a positive regulator of microglia-induced excessive neuroinflammation following ischemic stroke. Interventions targeting SIRT5 should therefore alleviate neuroinflammation and protect against ischemic stroke. Here, we synthesized a membrane-permeable peptide specifically bound to SIRT5 through a chaperone-mediated autophagy targeting motif (Tat-SIRT5-CTM) and examined its therapeutic effect in vitro and in vivo. First, in primary microglia, Tat-SIRT5-CTM suppressed the binding of SIRT5 with annexin-A1 (ANXA1), leading to SIRT5 degradation and thus inhibition of SIRT5-mediated desuccinylation of ANXA1, followed by increased membrane accumulation and secretion of ANXA1. These changes, in turn, alleviated microglia-induced neuroinflammation. Moreover, following intravenous injection, Tat-SIRT5-CTM could efficiently pass through the blood‒brain barrier. Importantly, systemic administration of Tat-SIRT5-CTM reduced the brain infarct area and neuronal loss, mitigated neurological deficit scores, and improved long-term neurological functions in a mouse model of ischemic stroke. Furthermore, no toxicity was observed when high doses Tat-SIRT5-CTM were injected into nonischemic mice. Collectively, our study reveals the promising efficacy of the peptide-directed lysosomal degradation of SIRT5 and suggests it as an effective therapeutic approach for the treatment of ischemic stroke.

Resveratrol Inhibited Nanoparticles Stromal Interaction Molecule 2 in Regulating miR-20b-5p Signaling Pathway to Improve Mitochondrial Function During Myocardial Ischemia-Reperfusion Injury

Myocardial ischemia-reperfusion (IR) in diabetes can cause severe myocardial damages. In this study, resveratrol (RES) nanoparticles were used in diabetic myocardial IR rat model injury to assess its effect on mitochondria function. Rat models were assigned into sham group, IR group, IR+RES group, IR+RES+mir-NC group, and IR+RES+miR-20b-5p inhibitor group. Myocardial infarction area was measured by TTC in 5 rats from each group, and ultrasound was used to detect left ventricular end-systolic internal diameters (LVIDs) and end-diastolic internal diameters (LVIDd), along with analysis of cardiomyopathy by HE staining. miR-20b-5p and Stromal interaction molecule 2 (STIM2) expressions, cardiomyocyte proliferation, apoptosis, cell viability, mitochondrial function, and relationship between miR-20b-5p and STIM2 were also analyzed. Resveratrol (RES) nanoparticles were prepared successfully. Myocardial infarct size, LVIDd and LVIDs of rats in IR+RES group decreased (vs. IR group), but were higher than sham group. miR-20b-5p expression also increased in the IR+RES group (vs. IR group), and the above indicators were decreased by the miR-20b-5p inhibitor (vs. IR+RES group, P <0.05). The myocardial changes in rats from the IR+RES+miR-20b-5p antagomir group were smaller (vs. IR group), while STIM2 expression was lower than in the IR group after using the RES nanoparticles (P < 0.05). RES nanoparticles can thus enhance mitochondrial function and cell viability of cardiomyocytes, increasing cell proliferation rate and decreasing apoptosis rate (vs. IR group,P <0.05). After using the RES nanoparticles to interfere with myocardial IR in the diabetic rats, they were found to inhibit STIM2 and improve mitochondria by regulating miR-20b-5p signaling pathway.

Endogenous Protein C Activation with AB002 Improves Outcomes in a Rat Model of Acute Ischemic Stroke

Background: The hemorrhagic side-effects of current thrombolytic agents limit their usefulness to a small subset of acute ischemic stroke patients. An early, effective, and hemostatically safe antithrombotic/neuroprotectant in addition to standard of care could improve outcomes. AB002 (E-WE thrombin) is an engineered recombinant thrombin analog (W215A/E217A) that activates cytoprotective and antithrombotic endogenous protein C on intravascular surfaces without any measurable procoagulant activity. In renal failure patients undergoing hemodialysis, AB002 treatment reduced dialyzer clotting without increasing markers of hemostasis impairment or coagulation (NCT03963895). Preclinical studies also showed that AB002 exhibits potent antithrombotic/thrombolytic effects in a non-human primate model of acute vascular graft thrombosis, as well as reduces infarct size and improves outcomes in an experimental acute myocardial ischemia-reperfusion model in mice. Aims: Here, we evaluated whether neuroprotective effects of AB002 can be observed following middle cerebral artery occlusion-recanalization (MCAO-R) in rats. Methods: The local animal care and use committee approved all studies. 8 to 9-week-old male rats (275-377 g) were anesthetized under isoflurane and a small laser-Doppler probe was affixed to the skull to monitor cortical perfusion and verify vascular occlusion and reperfusion. A silicone-coated 4-0 nylon monofilament was inserted into the right internal carotid artery (ICA) via the external carotid artery (ECA) until the laser-Doppler signal dropped to < 30 % of baseline. To evaluate the effect of AB002 on outcomes, rats (n = 4/ group) were treated with 25 µg/kg (0.25 mL/kg; i.v.) or saline placebo, administered 30 min after induction of ischemia. Since prolongation of the activated partial plasminogen time (aPTT) is a pharmacologic effect of high dose drug exposure, a 0.45 mL blood sample was collected at 20 min post treatment from the right jugular vein, processed to plasma and frozen for subsequent aPTT evaluation. At 90 min, the filament was removed, the surgical incisions were sutured closed and postoperative animals were monitored during recovery. Neurological performance scores and weight were assessed daily for 4 days. Rats were sacrificed on day 7 post-surgery and infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. To account for the effect of edema, the infarcted area was estimated indirectly by subtracting the non-infarcted area in the ipsilateral hemisphere from the contralateral hemisphere and expressing infarct volume as a percentage of contralateral hemisphere. APTT was measured in duplicate using a KC-4 coagulometer. All analyses were performed by researchers blinded to treatment. Results: Compared to placebo, rats that received AB002 exhibited less ischemic damage, evidenced by a statistically significant reduction in infarct volume of the cortex (32.3% versus 8.9%, P < 0.05) and hemisphere (28.0% versus 9.8%, P < 0.01). In addition, rats had better neurological scores and greater weight gains during recovery following treatment with AB002, compared to placebo. Mean aPTT values were higher in AB002 treated rats compared to placebo (34 ± 2.2 s versus 21 ± 1.6 s), confirming drug exposure. Conclusions: Treatment with AB002 during ischemia reduced infarct volume and improved outcomes in the rat experimental stroke model. Considering its neuroprotective and antithrombotic properties, we propose that early treatment with AB002 may confer outcome benefit for patients experiencing acute thrombotic emergencies.

A Novel Adenovirus (Adv)-Vaccinated Murine Model Simulates the Prothrombotic Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) Clinical Disorder

Background: VITT has emerged as a rare (10 -5 to 10 -6 vaccinated individuals), but life-threatening side-effect of post-AdV-based COVID-19 vaccination, characterized by rapidly progressive catastrophic thrombosis, thrombocytopenia and high levels of platelet-activating anti-platelet factor 4 (PF4) antibodies (Abs). While the disorder resembles heparin-induced thrombocytopenia (HIT) in that both anti-PF4 Abs activate platelets via FcγRIIA in the presence of PF4, the Abs differ in their requirements for heparin, i.e. HIT Abs require heparin, but VITT Abs do not. Moreover, alanine substitutions in PF4 revealed distinct antigenic sites in these two prothrombotic disorders. We observed that the defined VITT antigenic site, unlike the HIT antigenic site, is conserved on a second platelet-specific chemokine structurally similar to PF4, neutrophil activating peptide 2 (NAP2) and its precursor platelet-basic protein (PBP) and proposed that PBP as well as PF4 may be able to induce VITT. Objective: We have now generated a murine model of VITT (mVITT) to better understand the pathogenesis of this immune disorder and to examine whether PBP may be involved in this process. Methods: Transgenic FcγRIIA mice (n>5/group) that lack mPF4 (PF4 KO), but express human (h) PF4 were injected intramuscularly with 5x10 9 viral particles/mouse of ChadOx1-nCoV-19 (“nCoV-19 AdV”) leading to expression of the COVID-19 spike protein or control ChadOx-GFP, (“GFP AdV”) expressing the green fluorescent protein. To augment mVITT Ab production, mice also received CpG oligodeoxynucleotides (ODN 2395 or “ODN”, 10µg/mouse i.p for 5 days) as an adjuvant. In these latter experiments, ODN only- and carrier alone- (physiologic buffered saline, “PBS”) injected mice served as controls. The clinical course, blood counts and ELISA for Abs to spike protein, hPF4 and mPBP/NAP2 were followed once a week and organs were harvested for histology at the endpoint. Results: Only mice that received the nCoV-19 developed anti-spike protein Abs. In the studies without ODN, 1 out of the 20 mice injected with either nCoV-19 or GFP AdV developed progressive thrombocytopenia, massive thrombi in the lungs, heart and brain, and died at day 43, one day after receiving a second dose of GFP. This mouse developed a high titer of anti-mNAP2 Abs, but no significant increase in anti-hPF4 ( Table 1). In the studies with ODN adjuvant, 2 out of the 5 mice injected with nCoV-19 AdV and one of the 5 mice injected with GFP AdV developed thrombocytopenia and died at days 14, 39 and 40 after the 1 st dose and prior to being able to receive a second dose. Gross histology of the brains showed areas of infarction and/or hemorrhage. All three affected mice had raised anti-hPF4 Abs ( Table 1). Further characterization of these mice revealed newly formed anti-hPF4 Abs that were heparin independent. Moreover, two monoclonal (mo) Abs, a humanized version of KKO (G4 KKO) that only binds to the HIT antigenic site and 1E12 shown to bind to the VITT antigenic site, were tested for their ability to block the binding of mVITT Ab to hPF4. Only 1E12 blocked that binding (Figure 1). None of the ODN or PBS control mice developed thrombocytopenia or died ( Table 1). Conclusions: We have developed a murine model to simulate the development of VITT that supports AdV:chemokine complex formation as being key to the development of Abs and the prothrombotic state in VITT. This model indicates that anti-PBP/NAP2 Abs can develop after AdV immunization. We also show that the inclusion of an oligonucleotide adjuvant switched antigenic targeting to hPF4. The anti-hPF4 Ab did not develop when adjuvant alone was given, and the resultant Abs clearly bound as VITT-like Abs and not HIT Abs. The basis for the apparent ability of ODN to switch which chemokine is immunogenic will need future study. This model system may be useful in designing AdV that do not induce VITT as backbone for vaccines in the future as well.

Ion cocktail therapy for myocardial infarction by synergistic regulation of both structural and electrical remodeling

AbstractMyocardial infarction (MI) is a leading cause of death worldwide. Few drugs hold the ability to depress cardiac electrical and structural remodeling simultaneously after MI, which is crucial for the treatment of MI. The aim of this study is to investigate an effective therapy to improve both electrical and structural remodeling of the heart caused by MI. Here, an “ion cocktail therapy” is proposed to simultaneously reverse cardiac structural and electrical remodeling post‐MI in rats and minipigs by applying a unique combination of silicate, strontium (Sr) and copper (Cu) ions due to their specific regulatory effects on the behavior of the key cells involved in MI including angiogenesis of endothelial cells, M2 polarization of macrophages and apoptosis of cardiomyocyte. The results demonstrate that ion cocktail treatment attenuates structural remodeling post‐MI by ameliorating infarct size, promoting angiogenesis in both peri‐infarct and infarct areas. Meantime, to some extent, ion cocktail treatment reverses the deteriorative electrical remodeling by reducing the incidence rate of early/delayed afterdepolarizations and minimizing the heterogeneity of cardiac electrophysiology. This ion cocktail therapy reveals a new strategy to effectively treat MI with great clinical translation potential due to the high effectiveness and safety of the ion cocktail combination.

Evaluating the axonal injury and predicting the motor function recovery in supratentorial acute stroke patients

PurposeThis study aims to evaluate axonal injury in supratentorial acute stroke patients and predict motor function recovery. MethodsA cross-sectional descriptive study was performed on 28 patients with supratentorial acute stroke. All patients underwent brain magnetic resonance imaging (MRI) at Bach Mai Hospital from September 2021 to August 2022. Diffuse tensor imaging (DTI) was conducted using a 3-Tesla MRI machine to evaluate the association between the corticospinal tract and infarct area. Therefore, axonal injury and motor function recovery levels could be predicted. ResultsAlmost patients had no change in the signal of the axons, which did not pass through the infarct lesion (28.6%), and the signals strongly decreased in patients whose axons completely stayed inside the infarct lesion (32.1%). The fractional anisotropy (FA) and apparent diffusion coefficient (ADC) indices of the axons on the infarcted side were lower than those of axons on the contralateral side. The patient group, in which the axons did not pass through the infarct lesion or had no change in axonal signals, had a higher rate of better motor function recovery after 3 months than the other groups (39.3% and 25 %, respectively), whereas the axon group, in which the axons stayed completely inside the infarct lesion or had a remarkably decreased signal, had a very poor rate of recovery (32.1% and 39.3 %, respectively). The FA index of the axons on the infarct side in the poor recovery group was lower than that in the good recovery group. The ADC index did not differ between the groups. ConclusionMRI finding with axonal signal, location, and infarct side could significantly predict motor recovery after 3 months in acute stroke patients.

Gut microbiota-mediated ursodeoxycholic acids regulate the inflammation of microglia through TGR5 signaling after MCAO

Ischemic stroke has been demonstrated to cause an imbalance of gut microbiota. However, the change in gut microbiota-mediated bile acids (BAs) metabolites remains unclear. Here, we observed a decrease in gut microbiota-mediated BAs, especially ursodeoxycholic acid (UDCA), in the serum of stroke patients as well as in the intestine, serum and brain of stroke mice. Restoration of UDCA could decrease the area of infarction and improve the neurological function and cognitive function in mice in association with inhibition of NLRP3-related pro-inflammatory cytokines through TGR5/PKA pathway. Furthermore, knocking out TGR5 and inhibiting PKA activity reduce the protective effect of UDCA. Taken together, our results suggest that microbiota-mediated UDCA plays an important role in alleviating inflammatory responses and might be a promising therapeutic target in ischemic stroke.

Cardiac reprogramming reduces inflammatory macrophages and improves cardiac function in chronic myocardial infarction

Cardiomyocytes (CMs) have little regenerative capacity. After myocardial infarction (MI), scar formation and myocardial remodeling proceed in the infarct and non-infarct areas, respectively, leading to heart failure (HF). Prolonged activation of cardiac fibroblasts (CFs) and inflammatory cells may contribute to this process; however, therapies targeting these cell types remain lacking. Cardiac reprogramming converts CFs into induced CMs, reduces fibrosis, and improves cardiac function in chronic MI through the overexpression of Mef2c/Gata4/Tbx5/Hand2 (MGTH). However, whether cardiac reprogramming reduces inflammation in infarcted hearts remains unclear. Moreover, the mechanism through which MGTH overexpression in CFs affects inflammatory cells remains unknown. Here, we showed that inflammation persists in the myocardium until three months after MI, which can be reversed with cardiac reprogramming. Single-cell RNA sequencing demonstrated that CFs expressed pro-inflammatory genes and exhibited strong intercellular communication with inflammatory cells, including macrophages, in chronic MI. Cardiac reprogramming suppressed the inflammatory profiles of CFs and reduced the relative ratios and pro-inflammatory signatures of cardiac macrophages. Moreover, fluorescence-activated cell sorting analysis (FACS) revealed that cardiac reprogramming reduced the number of chemokine receptor type 2 (CCR2)-positive inflammatory macrophages in the non-infarct areas in chronic MI, thereby restoring myocardial remodeling. Thus, cardiac reprogramming reduced the number of inflammatory macrophages to exacerbate cardiac function after MI.

Developing and evaluating a chronic ischemic cardiomyopathy in swine model by rest and stress CMR.

A large animal model of chronic coronary artery disease (CAD) is crucial for the understanding the underlying pathophysiological processes of chronic CAD and consequences for cardiac structure and function. The goal of this study was to develop a chronic model of CAD in a swine model and to evaluate the changes of myocardial structure, myocardial motility, and myocardial viability during coronary stenosis. A total of 30 swine (including 24 experimental animals and 6 controls) were enrolled. The chronic ischemia model was constructed by using Ameroid constrictor in experimental group. The 24 experimental animals were further divided into 4 groups (6 animals in each group) and were sacrificed at 1, 2, 3 and 4 weeks after operation for pathological examination, respectively. Cardiac magnetic resonance (CMR) was performed preoperatively and weekly postoperatively until sacrificed both in experimental and control group. CMR cine images, rest/adenosine triphosphate (ATP) stress myocardial contrast perfusion and LGE were performed and analyzed. The rest wall thickening (WT) score was calculated from rest cine images. The MPRI (myocardial perfusion reserve index) and MPR (myocardial perfusion reserve) were calculated based on rest and stress perfusion images. Pathology staining including triphenyltetrazolium chloride, HE and picrosirus red staining were performed after swine were sacrificed and collagen volume fraction (CVF) was calculated. The time to formation of ischemic, hibernating, and infarcted myocardium was recorded. In experimental group, from 1w to 4w after surgery, the rest WT score decreased gradually from 35.2 ± 2.0%, 32.0 ± 2.9% to 30.5 ± 3.0% and finally 29.06 ± 1.78%, p < 0.001. Left ventricular ejection fraction was gradually impaired after modeling (58.9 ± 12.6%, 56.3 ± 10.1%, 55.3 ± 9.0%, 53.8 ± 9.9%, respectively). And the MPR and MPRI also decreased stepwise with extent of surgery time (MPRI dropped from 2.1 ± 0.4, 2.0 ± 0.2 to 1.8 ± 0.3 and finally 1.7 ± 0.1, p = 0.004; MPR dropped from 2.3 ± 0.4, 2.1 ± 0.2 to 1.9 ± 0.4 and finally 1.8 ± 0.1, p < 0.001). Stronger associations between MPR, MPRI and CVF were paralleled lower wall thickening scores in fibrosis-affected areas. The ischemic myocardium was first appeared in the first week after surgery (involving ten segments), hibernated myocardium was first appeared in the second week after surgery (involving seventeen segments). LGE was first appeared in eight swine in the third weeks after surgery (16 segments). At 4w after surgery, average 9.6g scar tissue was found among 6 swine. At the same time, histological analysis established the presence of fibrosis and ongoing apoptosis in the infarcted area. In conclusion, our study provided valuable insights into the pathophysiological processes of chronic CAD and its consequences for cardiac structure and function in a large animal model through combining myocardial motion and stress perfusion.

HSP90β regulates EAAT2 expression and participates in ischemia‑reperfusion injury in rats.

Cerebrovascular diseases (CVDs) have become a global public health problem and ischemia‑reperfusion injury, the major cause of neurological impairment exacerbation, is closely related to excitotoxicity. The present study aimed to investigate the effects of changes in heat shock protein (HSP)90β expression and verify whether HSP90β regulates EAAT2 expression in a cerebral ischemia‑reperfusion injury model. Healthy adult Sprague‑Dawley (SD) male rats were used to establish a control group, sham‑operated group, middle cerebral artery occlusion (MCAO) group, empty virus group and lentivirus group. A model of cerebral ischemia‑reperfusion was established using the MCAO method. Lentivirus construction and injection were used to interfere with the expression of HSP90β. The modified neurological severity score was used to assess neurological deficits. Triphenyltetrazolium chloride staining was used to detect infarct areas. Immunofluorescence was used to detect HSP90β expression localization and the expression levels of HSP90β and EAAT2 were determined using western blotting and reverse transcription‑quantitative PCR. An MCAO model was successfully established and it was found that HSP90β, but not HSP90α, was upregulated after MCAO. HSP90β expression coincided with astrocyte markers in the ischemic penumbra area, while no expression was observed in microglia. Inhibition of HSP90β expression improved neurological deficits and alleviated brain injury by increasing EAAT2 expression. These results suggested that HSP90β is involved in the process of cerebral ischemia‑reperfusion injury in rats and that inhibition of HSP90β expression increases EAAT2 levels, conferring a neuroprotective effect in MCAO model rats.

Improved Visualization and Quantification of Net Water Uptake in Recent Small Subcortical Infarcts in the Thalamus Using Computed Tomography.

Diagnosing recent small subcortical infarcts (RSSIs) via early computed tomography (CT) remains challenging. This study aimed to assess CT attenuation values (Hounsfield Units (HU)) and net water uptake (NWU) in RSSI and explore a postprocessing algorithm's potential to enhance thalamic RSSI detection. We examined non-contrast CT (NCCT) data from patients with confirmed thalamic RSSI on diffusion-weighted magnetic resonance imaging (DW-MRI) between January 2010 and October 2017. Co-registered DW-MRI and NCCT images enabled HU and NWU quantification in the infarct area compared to unaffected contralateral tissue. Results were categorized based on symptom onset to NCCT timing. Postprocessing using window optimization and frequency-selective non-linear blending (FSNLB) was applied, with interpretations by three blinded Neuroradiologists. The study included 34 patients (median age 70 years [IQR 63-76], 14 women). RSSI exhibited significantly reduced mean CT attenuation compared to unaffected thalamus (29.6 HU (±3.1) vs. 33.3 HU (±2.6); p < 0.01). Mean NWU in the infarct area increased from 6.4% (±7.2) at 0-6 h to 16.6% (±8.7) at 24-36 h post-symptom onset. Postprocessed NCCT using these HU values improved sensitivity for RSSI detection from 32% in unprocessed CT to 41% in FSNLB-optimized CT, with specificities ranging from 86% to 95%. In conclusion, CT attenuation values and NWU are discernible in thalamic RSSI up to 36 h post-symptom onset. Postprocessing techniques, particularly window optimization and FSNLB, moderately enhance RSSI detection.

ADAM8 deficiency in macrophages promotes angiogenesis to antagonize post-myocardial infarction cardiac injury

Abstract Background A disintegrin and metallopeptidase domain 8 (ADAM8) may be closely related to the progress of cardiovascular diseases. ADAM8 was first found in macrophages from mice, and ADAM family is closely related to the secretion function of macrophages. Therefore, ADAM8 may affect the process of myocardial infarction (MI) by regulating macrophage function. Purpose This study aims to explore how ADAM8 regulating the function of macrophages to inhibit the cardiac repair after MI. Methods CRISPR/Cas9 system was used to generate macrophage-specific ADAM8 knockout mice (ADAM8flox/flox,Lyz2-Cre, ADAM8 mKO) and ADAM8flox/flox (control) . Bone marrow transplantation was performed and macrophage-specific ADAM8 overexpressed adeno-associated virus (AAV6-CD68-Adam8) was generated. Proteomics of hearts and RNA-sequencing of bone marrow derived macrophages (BMDM) was performed. Cellular and molecular assays were used to discover the underlying mechanisms of ADAM8 function. Results (1) ADAM8 in serum of AMI patients was significantly higher than control group. ADAM8 was highly expressed in cardiac macrophages after day 3 of AMI. (2) Compared with the control group, the expression of CD31 and VEGFA in the infarcted myocardium was significantly increased in ADAM8 mKO mice, indicating enhanced angiogenesis. The cardiac fibrosis was decreased and heart function was improved in ADAM8 mKO group. ADAM8 overexpression can reverse the increase of angiogenesis caused by ADAM8 mKO and promote cardiac fibrosis in mice. The bone marrow transplantation experiments also showed that donor of ADAM8 mKO mice had increased angiogenesis and reduced cardiac fibrosis in the infarcted area. (3) The proteomics of hearts in AMI day 14, showed the Cardiac muscle contraction pathway and the Citrate cycle (TCA cycle) pathway in ADAM8 mKO mice were both increased significantly, indicating improved cardiac function and maintained the metabolic stability of heart after MI. Myocardial injection in situ of anti-VEGFA antibody can reverse the angiogenesis induced by ADAM8 mKO, and deteriorate heart function. (4) The supernatants and protein lysates of ADAM8 KO BMDM showed the decreased level of VEGFA, and IL-1β/IL-18. (5) RNA-sequencing showed that AMPK signaling pathway and autophagy pathway were both significantly increased in ADAM8 KO BMDM treated with LPS. p-AMPK/AMPK and autophagy-related molecules were obviously elevated in ADAM8 KO BMDM. Application of autophagy inhibitor 3-Methyladenine (3-MA) could reverse ADAM8 KO induced secretion of VEGFA and IL-1β/IL-18. Conclusions Our study has discovered ADAM8 as a new regulator of myocardial infarction. ADAM8 KO may promoting VEGFA secretion and inhibiting inflammatory factors secretion of macrophages via AMPK/autophagy pathway, further promoting angiogenesis and reducing fibrosis of infarcted myocardium, and attenuating heart function and adverse remodeling. ADAM8 may be a new target for treating myocardial infarction.Schematic diagram

Cardiac extracellular matrix at microvascular obstruction area after reperfused myocardial infarction: a morphometric study

Abstract Background Extracellular matrix (ECM), a structural and biomechanical support to cardiomyocytes, suffers substantial alterations after myocardial infarction (MI). Despite a complete reopening at epicardial level, hypoperfusion within the infarcted myocardium, known as microvascular obstruction (MVO), occurs. Extensive clinical studies and meta-analysis reported the association between the magnitude of MVO and the appearance of adverse remodeling; thus, scrutinizing ECM composition within the infarcted areas with impaired tissue-level reperfusion could provide fundamental insight regarding MVO pathophysiology. Purpose To histologically compare ECM composition within the infarct zone regarding the presence or absence of MVO using an experimental swine model of reperfused MI. Methods MI was induced in swine (n=10) by transitory 90-min coronary occlusion followed by 7 days of reperfusion. Prior to euthanasia, intracoronary thioflavin-S was infused. Within the infarcted myocardium, regions displaying MVO (thioflavin-S-) or no MVO (thioflavin-S+) were isolated and stained to morphometrically compare ECM composition. Results Regarding cellularity, areas with MVO displayed an enlarged presence of neutrophils and lymphocytes, whilst no differences in the amount of macrophages and myofibroblasts were detected in comparison to no MVO-myocardium. Those regions with macroscopic MVO showed lower capillary density than those without MVO. According to the fibers and ground substance, a significant reduction in the extension of type I collagen, laminin, and fibronectin together with an augmentation of glycosaminoglycan were noted in areas showing MVO compared to those without microvascular injury. No changes in the amount of type III collagen was observed. Conclusion The ECM composition in infarcted regions with MVO displays a higher presence of inflammatory infiltrate and glycosaminoglycans as well as a reduced amount of microvessels and fibers compared to those areas without microvascular hypoperfusion. These characteristics might underlie the development of adverse ventricular remodeling in MI patients with extensive MVO.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) as a novel regulator of post-infarction cardiac sympathetic hyperinnervation

Abstract Background Excessive outgrowth of sympathetic nerve fibers (sympathetic hyperinnervation) around the infarcted myocardium is associated with ventricular arrhythmia and an increased risk of sudden cardiac death. Post-infarction, epicardial cells exit quiescence through epithelial-to-mesenchymal transition (EMT), resulting in the formation of epicardium-derived cells (EPDCs). EPDCs support cardiac repair through differentiation into various cardiac cell lineages, as well as secretion of paracrine factors that stimulate processes such as angiogenesis. Recently, we demonstrated that human EPDCs reinforce neurite outgrowth in vitro. Whether EPDCs regulate cardiac innervation through paracrine signaling and contribute to post-infarction sympathetic hyperinnervation is unknown. Purpose To identify epicardial paracrine factors post-infarction that are involved in nerve growth. Methods Single-cell RNA-sequencing (scRNA-seq) datasets capturing epicardial cells of the healthy and infarcted adult mouse heart were collected from published research articles and were integrated into one object using Seurat. A list of gene products of known involvement in biological processes related to nerve growth (675 unique entries in total) was compiled. Based on this list, differential gene expression analysis on data from the integrated scRNA-seq object was performed to uncover highly-expressed candidates in the epicardial cell population. Epicardial expression of a candidate paracrine factor was validated by immunohistochemistry in the infarcted mouse heart as well as in an adult human in vitro model of EPDC EMT by immunocytochemistry and transcriptomic analysis. Results In our integrated scRNA-seq object, we identified high expression of Manf in epicardial cells post-infarction. Consistent with this finding, immunohistochemistry showed that Manf was up-regulated in the infarcted area, borderzone area, and epicardial region situated close to the infarct, an area where also hyperinnervation is observed. In vitro, MANF transcript expression was significantly down-regulated (p&amp;lt;0.01) in human EPDCs that were stimulated with TGFβ3 to induce EMT compared to EPDCs in which EMT was inhibited. In line with this, immunocytochemistry staining of MANF indicated that MANF expression was down-regulated in human EPDCs stimulated with TGFβ3. Conclusion(s) This study is the first to show that Manf is highly expressed in the epicardial region close to the infarct. In vitro, MANF is down-regulated in human adult EPDCs post-EMT. Future experiments will be directed toward determining the effect of MANF knockdown in human EPDC on neurite outgrowth in co-cultured human sympathetic neurons.MANF expression in human EPDCs after EMTManf expression after infarction

Mechanical LV support lowers diastolic LV pressure and prolongs diastolic coronary flow in the infarcted artery

Abstract Background Percutaneous left ventricular (LV) assist device provides not only circulatory support but also LV unloading. However, impact of LV unloading on coronary flow and mechanisms by which it is regulated remain unclear. We aimed to elucidate potential effects of LV unloading on coronary physiology in a pig model of myocardial infarction (MI). Methods First, six Yorkshire pigs underwent mechanical LV unloading using Impella two weeks after percutaneous MI induction. Regional blood flow (RBF) in infarct, border and remote myocardium were measured by fluorescent microsphere injections before and 120 min after LV unloading (Study 1). Next, we investigated coronary flow patterns with detailed temporal information. Six Yorkshire pigs were subjected to 90-minute balloon occlusion of the middle left anterior descending artery followed by reperfusion. At 60 minutes after reperfusion, LV unloading was initiated and maintained at maximal support. LV pressure, coronary flow and pressure in the infarcted artery were simultaneously recorded. Measurements were repeated with and without LV mechanical support. Coronary wave intensity was calculated from the flow/pressure data (Study 2). Results In Study 1, RBF calculated using microsphere was significantly increased only in the infarct area, but not in the border or remote areas. In Study 2, LV unloading resulted in lower LV pressure during the whole diastolic phase and higher coronary pressure during the late diastolic phase in all animals. Coronary flow velocity was higher throughout the diastole under LV unloading. In addition, the proportion of diastole to the whole cardiac cycle was significantly prolonged with LV unloading. Accordingly, area under the curve of coronary flow was increased during diastole and also for the whole cardiac cycle. In the wave intensity analysis, the magnitude of forward wave intensity was reduced by LV unloading, suggesting that the impact of forces from the aortic side was less significant under continuous device support. The duration between backward suction and pushing waves was extended, implicating earlier relaxation and delayed contraction, further supporting prolonged diastole from the myocardial force perspective. Conclusions Mechanical LV unloading increases coronary flow by prolonging diastolic phase in the infarcted artery.

Fibroblast-specific SOCS3 deficient mice exhibits decreased myocardial fibrosis and increased cardiomyocyte survival after acute myocardial infarction

Abstract Background Left ventricular (LV) remodeling after acute myocardial infarction (AMI) results in poor cardiac performance leading to heart failure. LV remodeling after AMI is characterized by infarct expansion, LV dilation, and fibrosis of viable myocardium. Suppressor of cytokine signaling-3 (SOCS3) is an endogenous negative-feedback regulator of the STAT3 signaling pathway. We previously reported that myocardial SOCS3 plays a detrimental role in LV remodeling after AMI; however, the role of STAT3 and SOCS3 within fibroblast in LV remodeling after AMI remains elusive. Objective The aim of this study is to clarify the role of STAT3 and SOCS3 within fibroblasts in LV remodeling during AMI in mice. Methods To investigate the role of STAT3 signaling and SOCS3 within fibroblast, we generated tamoxifen-induced fibroblast-specific SOCS3-deficient (fib-SOCS3 KO) mice. AMI was induced by permanent ligation of the left anterior descending artery in mice. After 2 weeks of induction with tamoxifen, histological evaluation by Hematoxylin Eosin staining and Picro-Sirius Red staining, cardiac function evaluation by echocardiography using a Vevo 3100 Echo Imaging System (Visual Sonics), and protein expression using Western blot analysis were performed. Results There was no significant difference in 2-week survival rate between fib-SOCS3 KO group and wild type (WT). Two weeks after AMI, ventricular weight was less in fib-SOCS3 KO mice compared to WT mice (p&amp;lt;0.05). In terms of cardiac function, LV ejection fraction was predominantly increased in fib-SOCS3 KO mice compared with WT mice, as measured by the Area method (p&amp;lt;0.05). There was no difference in LV end-diastolic area, but LV end-systolic area was significantly less in fib-SOCS3 KO mice than in WT mice. Histologically, residual viable myocardium in the infarcted area was greater in fib-SOCS3 KO mice compared with WT mice. The percentage of fibrotic area in the LV short-axis plane was significantly less in fib-SOCS3 KO mice than in WT mice. Western blot analysis revealed that phosphorylated-STAT3 tended to be elevated in fib-SOCS3 KO mice than in WT mice. Conclusions Our data suggest that STAT3 and SOCS3 within fibroblast may play an important role in the prevention of LV remodeling after AMI.

Recruited macrophages incorporate into the resident cardiac macrophage pool and converge into a common phenotype in the healing infarct, but not in the failing heart exposed to pressure overload

Abstract Introduction Resident macrophages account for 5% of the cells in the healthy heart. In response to cardiac injury, monocytes infiltrate and differentiate into recruited macrophages which function distinctly from the resident subset of primarily embryonic origin. In this work, we aim to identify the extent to which macrophage origin, tissue location and type of cardiac injury determine macrophage phenotypes in ischemic and non-ischemic cardiac injuries over time. Results and methods We use a tamoxifen inducible CX3CR1 Yfp CreER/+ :R26tdT/+ mouse line to visualize and quantify fluxes of resident and recruited macrophages, and their respective localizations within the heart following ischemia and reperfusion (I/R) injury and pressure overload after transversal aortic constriction (TAC), respectively. Interestingly, macrophage numbers peaked during the first week post-surgery in both the local/acute and the global/continuous injury models. Initially, recruited macrophages outnumbered the resident macrophage pool within the infarct area but ultimately reached a 1:1 equilibrium at 4 weeks post I/R injury and at 8 weeks post TAC. In I/R injury, the 1:1 equilibrium established already during the first week in the remote myocardium. Gene expression profiles changed alongside shifts in macrophage ratios. For instance, in the infarct, recruited macrophages showed upregulation of cytokine production and cell chemotaxis pathways at early stage. By 4 weeks post MI their gene expression profiles have converged with those of resident macrophages. Likewise, in the remote myocardium, recruited and resident macrophages assimilated post MI, overexpressing genes associated adaptative immune response activation and cellular homeostasis. Similar to the I/R injury setting, a 1:1 equilibrium of resident and recruited macrophages established in the hypertrophied and dilated hearts within 4 to 8 weeks post TAC surgery. However, transcriptional profiles remained differentially regulated between recruited and resident cardiac macrophages at all time points tested, and distinct from those observed post I/R injury: recruited macrophages behavior changed from a profibrotic into a proliferative phenotype, meanwhile resident macrophages featured prominently in tissue homeostasis. Conclusion Ischemic and non-ischemic, focal and global, acute and chronic cardiac injuries induce a transient peak of monocyte recruitment and macrophage differentiation in and around cardiac lesions which lead to a permanent integration of recruited monocyte-derived macrophages into the pool of tissue resident macrophages – albeit at different paces. The type of injury and macrophage localization within the remodeling tissue determine partial or complete override of ontogenic cell programs.

Changes in the immune cell landscape in the infarcted mouse heart

Abstract Background Myocardial Infarction (MI) and Ischemic Heart Failure represent the leading causes of death globally. Myocardial remodeling after MI is accompanied and driven by a strong immune response with an accumulation of several, partially opposing immune cell types. The temporal and spatial dynamics of this cardiac specific immune cell infiltration, however, have not been systematically evaluated. Purpose We characterized the dynamics of the immune cell repertoire in murine hearts following myocardial infarction by multi-color flow cytometry using a novel 12-marker panel allowing us to characterize major principal leukocyte lineages with a total of 10 distinct leukocyte populations. In parallel, we studied the immune response after MI in secondary lymph organs such as heart´s draining lymph nodes and the spleen. In addition, we performed transcriptional analysis of the healthy and infarcted hearts by bulk RNA at selected time points. Methods Permanent ligation of the left descending coronary artery (LAD), ischemic/reperfusion injury (I/R) and sham surgery was performed on C57BL6/J, 10 weeks old male mice. Leukocytes dynamics and T cell responses were screened in infarcted and remote areas of the heart, as well as in blood, spleen, and heat´s draining lymph nodes after 1, 3, 7, 14 and 28 days by multi-color flow cytometry. Also, immune activation and exhaustion transcription analysis by bulk RNA of healthy and infarcted hearts was performed at day 3,7 and 28 days after MI. Results We describe a sequential shift in the resident heart leukocyte population, a change in T cell activation states and identified potential new targets such as NK and γ/δ T cells. While macrophages area the most frequent leukocytes under steady state, making up to 75% of the total relative leukocyte population, at day 28 Lymphocytes represent 50%, among which B cells represent the most of them, followed by T helper cells. At early stages, CD4+ T cells secret pro inflammatory cytokines in the heart and in the heart´s draining lymph nodes, however, as stable scar in the heart is formed at day 7 immunomodulatory cytokines secretion such as IL-10 was observed. This is consistent with the build-up of a relevant adaptive immune response involving antigen presenting DCs and effector T and B cells in the healing heart. Conclusions Our findings contribute to identify novel cellular and transcriptional targets in the fight against ischemic heart failure.

The Presence of Communicating Arteries in the Circle of Willis Is Associated with Higher Rate of Functional Recovery after Anterior Circulation Ischemic Stroke.

Acute ischemic stroke (AIS) is the world's second leading cause of mortality. An established method for treating stroke patients in acute settings is endovascular therapy (EVT). However, the correlation of the successful endovascular treatment of AIS with the presence of communicating arteries in the circle of Willis needs to be proven. Our study examined clinical and radiological data of 158 consecutive patients treated with mechanical thrombectomy (MT) at our comprehensive stroke center. We analyzed their CT angiograms and digital subtraction angiography (DSA) to assess anatomical variants of Willis' circle and formed two groups-collateral-negative and collateral-positive group. The first group included patients with aplasia of both anterior (ACoA) and posterior communicating Artery (PCoA). The second group included patients that have at least one communicating artery (either anterior or posterior). We evaluated their reperfusion outcomes and functional recovery three months later. Our results showed that patients with communicating arteries had smaller areas of infarction on post-interventional CT and higher rates of functional recovery (Modified Rankin Score). The ACoA had a higher impact on early and late outcomes, confirmed by lower control CT scores and more favorable functional recovery. Therefore, anatomic variants of Willis' circle should be considered as a significant prognostic factor in AIS.