Cardiac Endothelial Cells Research Articles

Abstract 4137337: Targeting Macrophage NCOR1 to Alleviate Inflammation in Heart Failure with Preserved Ejection Fraction

Background: Inflammatory response is a key player in heart failure with preserved ejection fraction, however the underlying mechanisms remain unclear. Recent evidence indicates that macrophage inflammation driven by the transcriptional coregulatory protein NCOR1 could play a pivotal role in disease states. Hypothesis: NCOR1 orchestrates macrophage inflammation and cardiac dysfunction in HFpEF. Methods: HFpEF was induced in mice using a validated model combining high-fat diet and L-NAME for 15 weeks. Cardiac function was assessed using high-resolution ultrasound imaging and LV samples were used to study macrophage polarization and inflammation by FACS. We generated myeloid-specific NCOR1-KO mice to investigate the in vivo effects of macrophage NCOR1 depletion on HFpEF. In vitro experiments included RAW 264.7 cells polarization and the assessment of macrophage phenotype switching in the presence or the absence of NCOR1. To study the crosstalk of macrophages with the surrounding cardiac cells, cardiomyocytes (H9c2) and endothelial cells (HAECS) were exposed to conditioned media from cultured RAW. Results: The expression of macrophage markers in human LV specimens showed an increase in pro-inflammatory macrophages (M1) and a decrease in regulatory macrophages (M2) as compared to age-matched control mice. HFpEF mice showed increased expression of vascular adhesion molecules (ICAM1, ICAM2, E-selectin) and a significant recruitment of M1 macrophages as proved by qPCR, WB, and flow cytometry analysis. Of interest, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) – and lung congestion were significantly reduced in mice with macrophage NCOR1 deletion as compared to WT littermates, while exercise tolerance was significantly improved. These findings were associated with a reduction of myocardial inflammation (TNF-a, IL6, IL-1b) in Ncor1-KO mice. In vitro assays showed that stimulation of macrophages with PA induced M1 type switch and increased NCOR1, TNF-a, IL-6, and IL-1b levels, whereas NCOR1 depletion prevented detrimental changes of macrophage conditional medium (Fig.1). Conclusion: Targeting macrophage NCOR1 could represent a promising approach to blunt myocardial inflammation in HFpEF.

Single-Cell RNA Sequencing Uncovers Pathological Processes and Crucial Targets for Vascular Endothelial Injury in Diabetic Hearts.

Cardiovascular disease remains the leading cause of high mortality in individuals with diabetes mellitus. Endothelial injury is a major contributing factor for vascular dysfunction in diabetes. However, the precise mechanisms underlying endothelial cell injury and their heterogeneity in diabetes remains elusive. In this study, single-cell sequencing is performed in heart tissues from leptin receptor knock-out (db/db) diabetic mice at various pathological stages. Through cell cluster identification, differential gene analysis, intercellular communication analysis, pseudo time analysis, and transcription factor analysis, a novel mechanism of cardiac vascular endothelial damage in diabetes is identified. Specifically, a single-cell transcription map of cardiac vascular endothelial cells is presented in db/db mice. Diverse cellular clusters are found to play vital roles under diabetes-induced damage, highlighting crucial transcription factors involved in their regulation. In addition, the essential transcription factor Ets1 is found to protect against vascular endothelial injury in db/db mice. In summary, the work provides a comprehensive understanding of the development of diabetic cardiac vascular endothelial damage and the heterogeneity of the cells involved. These findings offer valuable insights into potential treatments and assessments of diabetic cardiovascular endothelial damage.

Pyroptosis is not likely to play a major role in anthracycline-induced cytotoxicity in H9c2 cardiomyocytes and human cardiac endothelial cells

Abstract Background Although anthracyclines are widely used as effective chemotherapeutic agents, anthracycline-induced cardiotoxicity (AIC) causes significant morbidity and mortality. The precise mechanism of AIC remains elusive, although apoptosis may be involved. Recent research suggests pyroptosis, a programmed lytic cell death pathway, might also contribute to AIC. Although studies have focused on AIC in the myocardium, emerging evidence suggests that AIC may also affect vascular cells. Furthermore, the cytotoxicity of doxorubicin (DOX) or its supposed active metabolite, doxorubicinol (DOXOL), has not been widely investigated in cardiac vascular cells. Purpose This study examines the hypothesis that pyroptosis plays a role in DOX and DOXOL cytotoxicity in H9c2 immortalised rat cardiac myoblasts and two types of human cardiac endothelial cells. Methods In vitro experiments were conducted to ascertain dose-dependent toxicity in H9c2, and cardiac endothelial cells following treatment with DOX or DOXOL. The type of cell death was determined by assessing cell morphology and fluorescent staining with markers of apoptosis (Annexin V) and lytic cell death (Propidium Iodide PI) following DOX or DOXOL treatment in comparison to known inducers of pyroptosis (LPS & Nigericin). Western blot experiments were conducted to study the expression of GSDMD (involved in pyroptosis), and caspase 3 (involved in apoptosis), following DOX or DOXOL. A human monocytic acute myeloid leukaemia cell line (THP-1 cells) was used as a positive control for pyroptosis. Results Dose-dependent cell death following DOX treatment was higher than with DOXOL in both H9c2 and cardiac endothelial cells. DOX caused apoptotic changes in all cell types. In contrast, DOXOL had weaker effects, inducing early apoptosis. No cleavage (activation) of GSDMD or caspase 3 was detected after DOX or DOXOL. Interestingly, in THP-1 cells, DOX induced predominantly apoptotic cell death, whereas DOXOL induced predominantly pyroptotic cell death, assessed both morphologically and by caspase 3 and GSDMD cleavage. Conclusion DOX and DOXOL primarily induce apoptotic cell death in H9c2 cardiomyocytes and cardiac endothelial cells. Interestingly, DOXOL, supposedly the active metabolite, was less cytotoxic than DOX. THP-1 cells undergo apoptotic or pyroptotic cell death with DOX or DOXOL, respectively. In conclusion, anthracycline-induced cytotoxicity is likely to be due to apoptotic injury rather than pyroptosis.

Empagliflozin treatment pre- and post- acute myocardial infarction reduces no-reflow, inflammatory cell infiltration and infarct size while preserving cardiac function.

Abstract Background and aims Empagliflozin (EMPA) and other members of the family of sodium glucose co-transporter 2 inhibitors have demonstrated clinical benefit in terms of survival and rehospitalizations in heart failure. However, their effect on acute myocardial infarction (AMI) is still under investigation. Microvascular injury (MVI) and no-reflow are major determinants of myocardial infarct size (IS) and prognosis. We investigated EMPA’s cardioprotective potential in AMI in terms of no-reflow, MVI and IS emphasizing on a translational regimen with EMPA treatment after AMI. Methods In a first series of experiments, C57Bl6 male mice (n=10 per group) were randomized into 1) Sham, 2) Control-AMI and 3) EMPA-Pre-AMI (10mg/kg/day orally for 6 weeks) groups. Mice underwent 30 min ischemia (I) and 2h reperfusion (R) or sham operation. Cell sorting and 3’ mRNA sequencing were applied to identify the cell types that are transcriptionally affected by EMPA pretreatment in mice with AMI. In a second series of experiments, the protocol was repeated, and an additional group was added, EMPA-Post-AMI (n=8-11 mice per group) in which oral treatment was performed 1h after R. Assessment of no-reflow via thioflavin S staining and electron microscopy was performed at 48 h. The infiltration of inflammatory cells in the ischemic heart was examined via flow cytometry. IS and myocardial function were determined by cardiac magnetic resonance (CMR). Type-2 diabetes mellitus patients received insulin (n=18) or EMPA (n=24) within 2 months post-AMI. Endothelial glycocalyx and endothelial-related circulating markers were examined at 4 months and 12 months post-AMI. Results EMPA pretreatment reversed AMI-induced alterations in cardiac endothelial cells’ (ECs) transcriptome. As such, it promoted the expression of survival genes, reduced gene expression related to adhesion molecules and ECs matrix degradation. Conversely, EMPA pretreatment exerted minimal effects on cardiomyocyte and fibroblast transcriptome. In support of the effect of EMPA on ECs, EMPA-Pre-AMI and EMPA-Post-AMI groups significantly reduced no-reflow and MVI as indicated by electron microscopy and histology at 48h of R. Moreover, both EMPA treatment pre-AMI and post-AMI reduced the infiltration of inflammatory monocytes and neutrophils in the infarcted myocardium suggesting enhanced vascular integrity. Both EMPA-Pre-AMI and EMPA-Post-AMI reduced IS and preserved cardiac function defined by CMR. Patients receiving EMPA presented with improved endothelial glycocalyx thickness, % global longitudinal strain and preserved pulse wave velocity. Conclusions EMPA treatment either before or after AMI protects against MVI, reduces no-reflow and IS and preserves cardiac function. Our data support the use of EMPA after recanalization in patients with AMI to confer improvement of cardiovascular function.

Soluble amyloid precursor protein-alpha mediates post-ischemic neovascularization after myocardial infarction

Abstract Background Amyloid precursor protein (APP) gives rise to amyloid-β peptides and thus has a key role in the pathogenesis of Alzheimer´s disease. APP is also highly expressed outside the brain including cells of the cardiovascular system, in particular endothelial cells (1). However, the role of APP and its proteolytic fragments in cardiovascular function and disease remains unknown. Purpose The main purpose of this study was to understand the function APP and the close homologue, amyloid precursor-like protein 2 (APLP2) in endothelial cells. Methods To test the potential role of endothelial APP and APLP2 in vivo, we generated inducible endothelium-specific APP/APLP2-double deficient mice (EC-APP/APLP2-KO) by crossing App fl/fl; Aplp2 fl/fl mice with the Cdh5-CreERT2 line. Animals were analyzed after exposure to myocardial infarction (MI). Human umbilical vein endothelial cells were used to study the mechanisms underlying APP/APLP2-mediated endothelial function in vitro. Results APP and APLP2 were found to be upregulated in cardiac endothelial cells after MI both in mice and humans. Loss of APP and APLP2 in endothelial cells had no effect on the basal heart function in mice. But, when subjected to MI, approximately 40 % of the EC-APP/APLP2-KO mice died within 3 weeks after MI in contrast to control animals, where hardly any death occurred. Survival of endothelium-specific APP- or APLP2-single knockouts was indistinguishable from that of control animals after MI. The surviving EC-APP/APLP2-KO mice showed larger infarct scars than the control animals. Magnetic resonance imaging and echocardiography at 3 weeks after MI revealed that EC-APP/APLP2-KO had more severe contractile dysfunction than the control mice (left ventricular ejection fraction 32.8 % vs. 44.9 %, respectively). As the underlying cause of this we identified a strong impairment of post-ischemia neovascularization in the absence of endothelial APP and APLP2. We found that soluble APP-α (APPsα) generated by non-amyloidogenic processing was the predominant form of APP in cardiac endothelial cells after MI. We therefore tested whether the expression of APPsα was able to rescue the observed phenotype of EC-APP/APLP2-KO after MI. Indeed, APPsα knock-in mice that solely express APPsα and no other APP fragments or cell surface APP showed the same phenotype as wild-type animals, indicating that APPsα is sufficient to mimic the function of APP. Under in vitro conditions, we found that APPsα was able to promote tube formation of cultured endothelial cells. APPsα also induced downstream signalling events such as AKT, ERK, and p38 activation which are reminiscent of growth factor induced signaling processes. Conclusions APPsα mediates post-ischemia neovascularization in the heart by exerting growth factor-like activity in endothelial cells, thereby protecting against cardiac injury.

Modulation of p300 acetylation to protect against doxorubicin-induced cardiotoxicity

Abstract Background Doxorubicin (DOX) is a widely-used anti-neoplastic agent, but the most common adverse reaction to its use is cardiotoxicity(1). DOX acts by producing reactive oxygen species (ROSs) that cause DNA damage(1,2), which activates various proteins, including the tumour suppressor p53 and the acetyltransferase p300. When activated, p300 acetylates and increases the activity of p53 which leads to increased apoptotic activity(3). Cardiotoxicity results because the heart has limited mechanisms to dispose of ROSs(4), and because cardiomyocytes have a low turnover rate(5). Purpose We examined the p300 inhibitor Theracurmin (THR)(6,7) as a candidate to prevent DOX cardiotoxicity using an in-vivo mouse model, and sought to elucidate the underlying molecular mechanisms using in-vitro studies. Methods C57BL/6 mice were pre-treated with THR or vehicle (as control), then administered DOX or vehicle. Cardiac function was evaluated by echocardiography and cardiac catheterization. In vitro studies used cardiac fibroblasts (FBs) and cardiac endothelial cells (ECs) pre-treated with THR, then with DOX, as in the animal studies. Western blotting and rt-qPCR was used to quantify protein and mRNA levels of genes in the p53-p300 pathway or related to apoptosis, oxidative stress, and cell cycle control. Results DOX-treated mice showed significant decreases in left ventricular (LV) ejection fraction (LVEF, p=0.0004), fractional shortening (FS, p=0.0012), and a significant increase in end-systolic volume (ESV, p=0.0004). We also showed a significant decrease in anterior (LVAW;d, p=0.005) and posterior (LVPW;d, p=0.005) wall thickness of the LV in these mice. However, when mice were pre-treated with THR prior to DOX administration, we saw significant increases in LVEF (p=0.045) and LVPW;d (p=0.015), and a significant decrease in ESV (p=0.0047) compared to those that were not pre-treated. In-vitro analyses demonstrated a significant increase in cleaved caspase 3 protein expression in DOX-treated ECs (p=0.007), that was not prevented by THR pre-treatment. In DOX-treated FBs, we saw a reduction in AKT mRNA (p=0.017) and increases in BAX 9p=0.008), P21 (p=0.0001), and GADD45 (0.037) mRNA expression. In DOX-treated ECs, we also saw significant increases in BAX (p=0.015) and P21 (p=0.011) mRNA expression. These changes in mRNA expression were not reversed with THR pre-treatment in either cell type. Conclusion THR pre-treatment successfully mitigated functional deficits and structural deficits associated with DOX cardiotoxicity. In-vitro studies show that THR pre-treatment was not sufficient to significantly prevent changes in key molecular targets associated with apoptosis. Further studies are required to elucidate the mechanism behind the observed functional changes.Abstract OverviewAbstract Results

Oxct1 regulates cardiomyocyte PANoptosis in acute myocardial infarction via succinylating Tfam

Abstract Background Myocardial ischemia leads to various forms of programmed cell death in cardiomyocytes, resulting in acute myocardial infarction (AMI) and the development of heart failure in the long term. PANoptosis is a unique cell death regulatory mechanism orchestrated by the PANoptosome complex, which regulates and intersects multiple forms of programmed cell death. Oxct1 has been demonstrated to play crucial roles in tumors, pressure-overloaded hearts, and cardiac endothelial cells. However, whether Oxct1 is involved in the regulation of PANoptosis and correlated mechanisms during AMI remains unclear. Purpose To elucidate the role of Oxct1 in cardiomyocytes during AMI and uncover the potential mechanisms. Methods The myocardial infarction model induced by anterior descending branch ligation and the primary cardiomyocytes hypoxia model were employed in our experiments. Small interfering RNA (siRNA) and adenovirus were constructed to knock down and overexpress Oxct1 for altering its expression in cardiomyocytes in vitro. Cardiomyocyte-specific knockout mice were constructed to alter Oxct1 expression in vivo. PANoptosis characteristic measurement, including Z-DNA binding protein 1(Zbp1), pyroptosis markers, apoptosis markers, necroptosis markers, ROS, and mitochondrial morphology, were performed to verify the ability of Oxct1 to regulate the PANoptosis of cardiomyocytes. Cardiac morphological, structural, and functional changes were measured to assess the effect of Oxct1 on AMI. RNA sequencing was performed to identify downstream pathways. Immunoprecipitation +Mass spectrometry and co-immunoprecipitation were performed to identify the specific binding factors of Oxct1. Results We found that the expression of Oxct1 was significantly elevated after AMI. Besides, gain- and loss-of-function experiments were constructed both in vitro and in vivo. Functionally, Oxct1 knockdown significantly enhanced cardiomyocyte vitality, reduced ROS production and mtDNA cytoplasmic release in hypoxic cardiomyocytes. In vivo, Oxct1cko mice manifested reduced infarct size, improved cardiac function and alleviated cardiac fibrosis by mitigating PANoptosis. However, Oxct1 overexpression resulted in the opposite effects. Afterwards, RNA-seq analysis results showed that cytosolic DNA-sensing pathway was significantly enriched in Oxct1 knockdown cardiomyocytes. Mechanistically, Oxct1 could bind to mitochondrial transcription factor A(Tfam) and increased its succinylation, promoting Tfam degradation. Downregulation of Tfam resulted in ROS production and mtDNA damage, while mtDNA released into cytoplasm could activate Zbp1 and induce cascade reaction of PANoptosis. Conclusion This study demonstrates that Oxct1 promotes Tfam degradation through succinylation, exacerbating ROS production, mtDNA damage and cytoplasmic release, ultimately leading to cardiomyocyte PANoptosis in AMI.

Human epicardial adipose tissue secretome alters metabolomic profile and induces endothelial-to-mesenchymal transition in human lymphatic endothelial cells

Abstract Background Cardiac lymphatic systems play an important role in maintaining myocardial fluid homeostasis during inflammation. We have recently reported the epicardial adipose tissue (EAT)-derived factor induces inflammation and fibrosis in atrial myocardium, which lead to atrial fibrillation (AF). However, the direct effect of EAT on the cardiac lymphatic endothelial cells (LECs) remains unknown. Objective To elucidate the effect of EAT on cardiac LECs, as one of the potential causes of AF. Methods Human EAT samples were collected from 18 consecutive patients with or without AF patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=9, 72.7±8.6 years) and the AF group (n=9, 73.3±7.4 years). Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation to mature adipocytes, cell culture supernatant was collected, and cultured with human LECs. Cell permeability was measured by using FITC-dextran assay. Tube forming ability was analyzed by seeding LECs on the extracellular matrix gels. Metabolomic profiling of LECs was performed by GC-MS system. Results RT-PCR analysis revealed that cell culture supernatant from mature adipocytes of AF patients significantly increased mRNA expression of mesenchymal marker, TAGLN/SM22a (p<0.01) in human LECs, whereas mRNA expression of lymphatic endothelial markers, LYVE1 (p=0.0552) and PROX1 (p=0.0231) were decreased in human LECs, compared to those from mature adipocytes of SR patients. These data suggest cell culture supernatant from mature adipocytes of AF patients highly induce endothelial-to-mesenchymal transition (EndMT) of LECs. When assessing the endothelial barrier function by using FITC-dextran, cell culture supernatant from mature adipocytes of AF patients significantly decreased cell barrier function (p<0.01) compared to those from mature adipocytes of SR patients. Furthermore, cell culture supernatant from mature adipocytes of AF patients significantly decreased the tube forming ability of LECs (p<0.01) compared to those from mature adipocytes of SR patients. To assess the metabolomic changes in human LECs by EAT-secretome, we conducted the GC/MS analysis. Cell culture supernatant from mature adipocytes of AF patients significantly decreased several amino acids including branched-chain amino acid (BCAA; valine, leucine and isoleucine), compared to those from mature adipocytes of SR patients. Conclusion Our study with human EAT and LECs demonstrated that EAT-secretome from AF patients altered metabolomic profile in LECs, and highly caused EndMT in LECs, leading to impaired barrier function and the tube forming ability. Defects of lymphangiogenesis could be a therapeutic target for atrial cardiomyopathy.

Resolving the developmental mechanisms of cardiac microthrombosis of SARS-CoV-2 based on single-cell transcriptome analysis.

The coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) developed into a global health emergency. Systemic microthrombus caused by SARS-CoV-2 infection is a common complication in patients with COVID-19. Cardiac microthrombosis as a complication of SARS-CoV-2 infection is the primary cause of cardiac injury and death in patietns with severe COVID-19. In this study, we performed single-cell sequencing analysis of the right ventricular free wall tissue from healthy donors, patients who died during the hypercoagulable period of characteristic coagulation abnormality (CAC), and patients who died during the fibrinolytic period of CAC. We collected 61,187 cells enriched in 24 immune cell subsets and 13 cardiac-resident cell subsets. We found that in the course of SARS-CoV-2 infected heart microthrombus, MYO1EhighRASGEF1Bhighmonocyte-derived macrophages promoted hyperactivation of the immune system and initiated the extrinsic coagulation pathway by activating chemokines CCL3, CCL5. This series of events is the main cause of cardiac microthrombi following SARS-CoV-2 infection. In a SARS-CoV-2 infected heart microthrombus, excessive immune activation is accompanied by an increase in cellular iron content, which in turn promotes oxidative stress and intensifies intercellular competition. This induces cells to alter their metabolic environment, resulting in increased sugar uptake via the glycosaminoglycan synthesis pathway. In addition, high levels of reactive oxygen species generated by elevated iron levels promote increased endogenous malondialdehyde synthesis in a subpopulation of cardiac endothelial cells. This exacerbates endothelial cell dysfunction and exacerbates the coagulopathy process.

12489 Senolytics Dasatinib And Quercetin Alleviate Type 1 Diabetes-Related Cardiac Fibrosis In Male Akita Ins-/+Mice

Abstract Disclosure: N. Kadoumi: None. G. Zatarah: None. J. Stokar: None. P. Shivam: None. I. Gurt: None. R. Dresner-Pollak: None. People living with type 1 diabetes (T1D) have a markedly shorter life expectancy compared to non-diabetic age- and sex-matched controls primarily due to cardiovascular disease. Diabetic cardiomyopathy is characterized by structural and functional alterations that occur independently of hypertension or coronary artery leading to congestive heart failure. The pathogenesis of diabetic cardiomyopathy involves endothelial cell dysfunction, microangiopathy, mitochondrial dysfunction, and oxidative stress, resulting in inflammation, extensive remodeling, cardiomyocyte hypertrophy, interstitial fibrosis, and apoptosis. T1D is associated with accelerated aging. Cellular senescence is a hallmark of aging, it is characterized by cell cycle arrest, distinct morphological alterations and the release of molecules referred to as the senescence-associated secretory phenotype (SASP). Senescent cardiomyocytes secrete SASPs that promote activation of fibroblasts, hypertrophy of cardiac muscle cells, and dysfunction of cardiac endothelial cells. An increase in cardiac cellular senescence was demonstrated in mouse models of T1D. Senolytics are compounds that clear senescent cells. Senolytics were shown to improve multiple outcomes in age-associated conditions in mice and humans.We investigated the effects of senolytics Dasatinib (D) and Quercetin (Q) on cardiac fibrosis in the AKITA Ins-/+ mouse model of T1D.We first assessed if there is increased collagen type1 expression in the heart ventricles and perivascular in 3-month-old in AKITA Ins-/+ vs. WT male mice using Immunofluorescence. Increased perivascular and interstitial expression of collagen 1 was found in AKITA Ins-/+ compared to WT age-and sex-matched mice (0.0811±0.008177 µm2 vs. 0.0533± 0.007960 µm2 ,P=0.0476), (105.7±10.29 µm2 vs. 54.85±14.61 µm2, P=0.0476), respectively. Next, 3-month-old AKITA Ins-/+wmale mice were randomly assigned to treatment with Dasatinib (5 mg/kg) and Quercetin (50 mg/kg) or vehicle administered once monthly by oral gavage for a total of four months (n=20, 10 in each group). Heart samples were analyzed for gene expression, collagen 1 protein by western blotting and Immunofluorescence and analyzed using QuPath 0.5.0 Software. Interstitial and perivascular fibrosis was separately assessed in the right and left ventricles. Treatment with D+Q resulted in a 2-fold decrease in COL1A1 gene and Collagen 1 protein expression by RT-PCR and western blot analysis (P= 0.011 and 0.042, respectively) in AKITA Ins-/+ mice. Immunostaining analysis revealed reduced Collagen 1 deposition in both the left and right ventricles (105.7±10.29 µm2 vs. 43.53±16.31 µm2, P=0.0571), as well as in the perivascular area (0.0811±0.008177 µm2 vs. 0.052±0.006163 µm2, P=0.1143). Treatment with senolytics D+Q reduces the expression of Collagen I, a key factor in myocardial fibrosis, in adult male AKITA Ins-/+ mice. Presentation: 6/3/2024

Selenoprotein M protects cardiac endothelial cell integrity against high-glucose stress via enhancing Parkin-mediated mitophagy

Selenoprotein M (SELENOM) has emerged as a crucial factor in maintaining cellular redox homeostasis and mitigating oxidative damage. This study aims to investigate its protective role in cardiac endothelial cells under hyperglycemic stress, a condition commonly associated with diabetes mellitus and its cardiovascular complications. Diabetic mice model and human umbilical vein endothelial cells (HUVECs) were applied for in vivo and in vitro studies. Results reveal that hyperglycemia significantly downregulates SELENOM expression in both diabetic mouse hearts and primary cultured cardiac endothelial cells. Overexpression of SELENOM in HUVECs mitigated high-glucose-induced FITC-Dextran diffusion and the loss of transendothelial electrical resistance. Additionally, SELENOM overexpression decreased reactive oxygen species (ROS) levels, preserved tight junction protein expression, and maintained cellular structural integrity under hyperglycemic conditions. Furthermore, SELENOM overexpression attenuated high-glucose-induced mitochondrial apoptosis. High-glucose conditions decreased Parkin and increased p62 and Beclin1 expressions. SELENOM overexpression restored Parkin levels and promoted co-localization of LAMP1 and TOMM20. Knockdown of Parkin significantly attenuated these protective effects, suggesting the importance of Parkin in Selenoprotein M-mediated mitophagy. Collectively, these findings suggest that Selenoprotein M enhances Parkin-mediated mitophagy to protect endothelial cells from hyperglycemic stress, offering potential therapeutic insights for diabetic cardiovascular complications.

Deubiquitinase USP5 regulates RIPK1 driven pyroptosis in response to myocardial ischemic reperfusion injury

BackgroundGasdermin D (GSDMD) mediated pyroptosis plays a significant role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. However, the precise mechanisms regulating pyroptosis remain unclear. In the study, we aimed to investigate the underlying mechanism of pyroptosis in myocardial I/R injury.MethodsIn the present study, we analyzed the effects of USP5 on the RIPK1 kinase activity mediated pyroptosis in vitro after H/R (hypoxia/reoxygenation) and in vivo in a MI/R mouse model. TTC and Evan’s blue dye, Thioflavin S and immunohistochemistry staining were performed in wild-type, RIPK1flox/flox Cdh5-Cre and USP5 deficiency mice. CMEC cells were transfected with si-USP5. HEK293T cells were transfected with USP5 and RIPK1 overexpression plasmid or its mutants. The levels of USP5, RIPK1, Caspase-8, FADD and GSDMD were determined by Western blot. Protein interactions were evaluated by immunoprecipitation. The protein colocalization in cells was monitored using a confocal microscope.ResultsIn this study, our data demonstrate that RIPK1 is essential for limiting cardiac endothelial cell (CMEC) pyroptosis mediated by caspase-8 in response to myocardial I/R. Additionally, we investigate the role of ubiquitin-specific protease 5 (USP5) as a deubiquitinase for RIPK1. Mechanistically, USP5 interacts with RIPK1, leading to its deubiquitination and stabilization.ConclusionsThese findings offer new insights into the role of USP5 in regulating RIPK1-induced pyroptosis.

Ulinastatin attenuated cardiac ischaemia/reperfusion injury by suppressing activation of the tissue kallikrein-kinin system.

Ulinastatin has beneficial effects in patients undergoing coronary artery bypass grafting (CABG) surgery due to its anti-inflammatory properties, but the underlying mechanism remains unclear. We used samples from patients undergoing CABG, a model of cardiac ischaemia-reperfusion injury (IRI) in mice and murine cardiac endothelial cell cultures to investigate links between ulinastatin, the kallikrein-kinin system (KKS), endothelial dysfunction and cardiac inflammation in the response to ischaemia/reperfusion injury (IRI). These links were assessed using clinical investigations, in vitro and in vivo experiments and RNA sequencing analysis. Ulinastatin inhibited the activity of tissue kallikrein, a key enzyme of the KKS, at 24 h after CABG surgery, which was verified in our murine cardiac ischaemia-reperfusion model. Under normal conditions, ulinastatin only inhibited kallikrein activity but did not affect bradykinin (B1/B2) receptors. Ulinastatin protected against IRI, in vivo and in vitro, by suppressing activation of the kallikrein-kinin system and down-regulating B1/B2 receptor-related signalling pathways including ERK/ iNOS, which resulted in enhanced endothelial barrier function, mitigation of inflammation and oedema, decreased infarct size, improved cardiac function and decreased mortality. Inhibition of kallikrein and knockdown of B1, but not B2 receptors prevented ERK translocation into the nucleus, reducing reperfusion-induced injury in murine cardiac endothelial cells. Treatment with ulinastatin exerts a protective influence on cardiac reperfusion by suppressing activation of the kallikrein-kinin system. Our findings highlight the potential of targeting kallikrein /bradykinin receptors to alleviate endothelial dysfunction, thus improving cardiac IRI.

Differential restriction of chikungunya virus in primary human cardiac endothelial cells occurs at multiple steps in the viral life cycle.

Arthropod-borne viruses (arboviruses) constitute a significant ongoing public health threat, as the mechanisms of pathogenesis remain incompletely understood. Cardiovascular symptomatology is emerging as an important manifestation of arboviral infection. We have recently studied the cardiac tropism and mechanisms implicated in cardiac damage in mice for the alphavirus chikungunya virus (CHIKV), and we therefore sought to evaluate the cardiac tropism of other emerging alphaviruses and arboviruses. Using human primary cardiac cells, we found that arboviruses from diverse viral families were able to replicate within these cells. Interestingly, we noted that while the closely related alphavirus Mayaro virus (MAYV) could replicate to high titers in primary human cardiac microvascular endothelial cells, pulmonary, and brain endothelial cells, the Indian Ocean Lineage of CHIKV (CHIKV-IOL) was completely restricted in all endothelial cells tested. Upon further investigation, we discovered that this restriction occurs at both entry and egress stages. Additionally, we observed that compared to CHIKV, MAYV may antagonize or evade the innate immune response more efficiently in human cardiac endothelial cells to increase infection. Overall, this study explores the tropism of arboviruses in human primary cardiac cells and characterizes the strain-specific restriction of CHIKV-IOL in human endothelial cells. Further work is needed to understand how the differential restriction of alphaviruses in human endothelial cells impacts pathogenesis in a living model, as well as the specific host factors responsible.

Endothelial α1-adrenergic receptor activation improves cardiac function in septic mice via PKC-ERK/p38MAPK signaling pathway

Cardiomyopathy is particularly common in septic patients. Our previous studies have shown that activation of the alpha 1 adrenergic receptor (α1-AR) on cardiomyocytes inhibits sepsis-induced myocardial dysfunction. However, the role of cardiac endothelial α1-AR in septic cardiomyopathy has not been determined. Here, we identified α1-AR expression in mouse and human endothelial cells and showed that activation of α1-AR with phenylephrine (PE) improved cardiac function and survival by preventing cardiac endothelial injury in septic mice. Mechanistically, activating α1-AR with PE decreased the expression of ICAM-1, VCAM-1, iNOS, E-selectin, and p-p38MAPK, while promoting PKC and ERK1/2 phosphorylation in LPS-treated endothelial cells. These effects were abolished by a PKC inhibitor or α1-AR antagonist. PE also reduced p65 nuclear translocation, but this suppression is not blocked by PKC inhibition. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on p38MAPK phosphorylation. Our results demonstrate that cardiac endothelial α1-AR activation prevents sepsis-induced myocardial dysfunction in mice by inhibiting the endothelial injury via PKC-ERK/p38MAPK signaling pathway and a PKC-independent inhibition of p65 nuclear translocation. These findings offer a new perspective for septic patients with cardiac dysfunction by inhibiting cardiac endothelial cell injury through α1-AR activation.

Abstract Tu004: Global distribution of cardiac exosomes after myocardial infarction

Background: Exosomes’ beneficial effects on the injured hearts show promising results. Understanding the biodistribution and kinetics of cardiac exosomes during myocardial infarction (MI) is crucial for exploring the underlying mechanisms. Aim: We aimed to elucidate dynamic biodistributions of cardiomyocyte-released exosomes in MI hearts at post-MI day 3 and day 14, representing the acute and remodeling stages, respectively. We employed the cardiac exosome reporter mice expressing CD63NanoLuc to track the endogenous cardiac exosomes. Method: The NanoLuc reporter mice underwent MI surgery (n=3-12/group). Reporter expression was induced by tamoxifen injection. To assess post-MI cardiac exosome distribution via circulation, fourteen organs, and plasma exosomes were harvested for luciferase activity quantification at each time point. Bioluminescence analysis was conducted in isolated cardiac fibroblasts, endothelial cells, and leukocytes to profile exosome-mediated paracrine signaling. Result: Plasma exosome luciferase activities were consistent across groups. A decline in bioluminescent levels was observed in all organs at both time points, with significant differences in the pancreas, thyroid, kidney, lung, and thymus (p<0.05). Day 3 post-MI showed increased heart endothelial cell bioluminescence in risk and remote areas (p=0.0007), whereas day 14 showed decreased bioluminescence in cardiac fibroblasts in both areas (p=0.0029 and p=0.0086). Endothelial cells and leukocytes exhibited a ten-fold higher exosome uptake than cardiac fibroblasts. Conclusion: There was a significant decrease in exosome uptake by major organs from the injured heart. Cardiac endothelial cells and fibroblasts demonstrated dynamic exosome uptake changes, with endothelial cells and leukocytes showing notably higher uptake compared to fibroblasts.

Abstract Mo075: Cigarette smoking causes somatic mutations in cardiac endothelial cells

Cigarette smoking (CS) is a major contributor to cardiovascular diseases (CVDs), responsible for one in every four CVD-related deaths. Despite this, the precise mechanisms through which smoking induces CVDs remain elusive. The chemicals in cigarette smoke, particularly oxidants, are known to instigate oxidative stress and activate the vessel wall. Alongside the dysregulation of cellular functions, oxidative stress can lead to DNA damage. Accumulation of DNA damage and somatic mutations is a recognized hallmark of aging, a significant risk factor for various age-related diseases, including CVDs, neurodegenerative diseases, and cancer. Recent studies have indicated a substantial increase in somatic mutations, with mutational signatures reflective of oxidative stress, in bronchial epithelial cells from smokers. Given their proximity in the blood circulation, these pathophysiological findings could naturally extend to the coronary system. Therefore, we hypothesized that CS induces somatic mutations and injuries in cardiac endothelial cells (ECs), laying the mechanisms for cigarette smoking-induced CVDs. To test this hypothesis, we conducted single-cell whole-genome sequencing (scWGS) on 105 cardiac endothelial cells isolated from postmortem hearts of 6 normal individuals, 3 smokers, and 4 smokers with atherosclerotic cardiovascular disease (ASCVD). Our results revealed a significant increase in somatic single nucleotide variants (SNVs) and insertions and deletions (indels) in cardiac ECs from smokers and those with ASCVD. These excess somatic mutations exhibited Catalogue Of Somatic Mutations In Cancer (COSMIC) mutational signatures, including SBS4, SBS29, SBS40, SBS92, and ID3, indicative of DNA damage and mutagenesis associated with cigarette smoking, oxidative stress, and formaldehyde exposure. Further analysis of single-nucleus RNA sequencing (snRNA-seq) data from postmortem hearts of 3 normal individuals, 4 smokers, and 1 smoker with ASCVD demonstrated upregulated pathways involved in apoptosis, EC migration, and vascular development. In conclusion, our study demonstrates that chemicals in cigarette smoke exert direct genotoxic effects on cardiac ECs, mirroring their impact on bronchial epithelial cells. Cigarette smoking significantly accelerates the accumulation of somatic mutations in cardiac ECs, resulting in apoptosis and vascular damage. Consequently, this damage leads to a secondary activation of angiogenesis for vascular repair.

Abstract Tu046: Mechanisms of Epicardium-Directed Cardiac Repair

Background: In the adult heart, the epicardium becomes activated by myocardial infarction (MI), contributing to cardiac remodeling primarily through the secretion of paracrine factors. However, there is a limited understanding of the cellular and molecular mechanisms that govern epicardial function and promote pro-reparative signaling pathways. Thus, enhancing or restoring the pro-regenerative characteristics of the epicardium may benefit cardiac repair after MI. We hypothesize that epicardial-derived cells preserve cardiac function during ischemic injury through the limited secretion of pro-reparative paracrine factors, such as slit guidance ligand 2 (Slit2), which enhances angiogenesis and vascular stability. Methods: We used the Wt1 (Wilms Tumor 1) CreERT2 ; R26 tdTomato mouse line for tamoxifen-inducible epicardial-specific lineage tracing and Pdgfra nGFP to label cardiac fibroblasts ( Wt1 CreERT2 ; R26 tdT ; Pdgfra nGFP ). Tagged epicardial-derived cells (tdTomato + ) from sham and 7-day post-MI mice were isolated by fluorescence-activated cell sorting and sequenced by single-cell RNA sequencing. To delineate Slit2-directed cell and molecular mechanisms, we performed in vitro studies and utilized adeno-associated viral vectors for gene therapy in a mouse model of MI. Results: We identified 9 transcriptionally distinct epicardial subpopulations contributing to paracrine signaling induced by MI. We found enriched expression of various chemokines in epicardial-derived stromal cells, while genes associated with Wnt signaling and epithelial-to-mesenchymal transition (EMT) were highly expressed in Wt1 -expressing clusters. Isolated cardiac fibroblasts treated with an adenoviral vector expressing Slit2 exhibited no differences in myofibroblast formation or collagen production following treatment with activation stimuli (TGFβ1/Ang II) but showed significant increases in the expression of pro-angiogenic gene programs. Cardiac-targeted AAV9-Slit2 overexpression in mice subjected to MI led to a striking increase in cardiac ejection fraction, expression of angiogenic-modulating genes, vessel density, and endothelial cell proliferation but did not alter fibrotic deposition. Conclusions: Our study reveals the dynamic role of epicardial cells during the post-MI cardiac repair process and delineates a Slit2-Robo dependent mechanism governing cardiac fibroblast and endothelial cell crosstalk to modulate angiogenesis.

Abstract We138: Single-nucleus and bulk multiomic analyses reveal the dynamics of individual cardiac cell types after myocardial infarction and the underlying gene regulatory networks

Cardiovascular diseases are the leading causes of death in the world, among which myocardial infarction (MI) is the most fatal form. Following MI, the death of cardiomyocytes induces cardiac remodeling, which is mainly mediated by cardiac fibroblasts but also involves other cardiac cell types. A deeper understanding of the dynamics of individual cell types among different stages after myocardial infarction may help identify novel therapeutic strategies. Here, through single-nucleus multiomic (snRNAseq and snATACseq) analysis of uninjured mouse hearts and mouse hearts at different time points (2 h, 1 d, 3 d, 7 d, and 4 w) after MI, we identified multiple cardiac cell types, including cardiomyocyte, fibroblast, epicardial cell, endothelial cell, mural cell, and immune cell, and their dynamic gene expression and chromatin accessibility after MI. Features specific to the acute and chronic responses of different cell types to MI were identified. Cell type-specific single-nucleus gene regulatory networks (snGRNs) constructed through integrated analysis of snRNAseq and snATACseq data revealed transcription factors possibly responsible for the gene expression changes in individual cell types. The cardiac fibroblast GRN was further enriched using bulk RNAseq and ATACseq data collected at more time points (uninjured, and 2 h, 12 h, 1 d, 3 d, 7 d, 2 w, and 4 w after MI) and non-coding RNA transcriptomics. Promoter and enhancer activities and chromatin interactions in cardiac fibroblasts were further verified by CUT&Tag targeting corresponding histone markers and Hi-C, respectively. The refined cardiac fibroblast GRN identified multiple key transcription factors that likely govern cardiac fibroblast activation and differentiation after MI.

Endothelial-to-mesenchymal transition enhances permissiveness to AAV vectors in cardiac endothelial cells

A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for invivo gene delivery. We screened a library of >1,500US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, asdocumented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. Invivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart.