Case report: heart failure related to intravitreal injection of anti-VEGF.

BACKGROUND: Intravitreal injection of anti-vascular endothelial growth factor is considered the first-line treatment for polypoidal choroidal vasculopathy. It has potential risks for circulatory system, which should be particularly carefully evaluated in older patients. In this case study, we aim to discuss the potential impact of this treatment regimen on cardiac health. CASE PRESENTATION: This case report describes an elderly patient with no prior history of heart disease who exhibited unexpected heart enlargement and dysfunction. Throughout the patient's hospital stay, various potential causes were investigated, leading to the hypothesis that a 10-year history of intravitreal injections of anti-vascular endothelial growth factor could be related to the observed clinical manifestations. The patient was advised to discontinue this treatment, and after a 2-month follow-up period, there was a gradual improvement in the patient's cardiac structure and function. CONCLUSION: This manuscript highlights the importance of conducting cardiac examinations before and after anti-vascular endothelial growth factor treatment, especially for individuals at risk of heart diseases like the elderly. It emphasizes the need to carefully weigh the benefits and risks of treatment regimens to ensure optimal therapeutic outcomes.

NKRF in Cardiac Fibroblasts Protects against Cardiac Remodeling Post-Myocardial Infarction via Human Antigen R.

Myocardial infarction (MI) remains the leading cause of death worldwide. Cardiac fibroblasts (CFs) are abundant in the heart and are responsible for cardiac repair post-MI. NF-κB-repressing factor (NKRF) plays a significant role in the transcriptional inhibition of various specific genes. However, the NKRF action mechanism in CFs remains unclear in cardiac repair post-MI. This study investigates the NKRF mechanism in cardiac remodeling and dysfunction post-MI by establishing a CF-specific NKRF-knockout (NKRF-CKO) mouse model. NKRF expression is downregulated in CFs in response to pathological cardiac remodeling in vivo and TNF-α in vitro. NKRF-CKO mice demonstrate worse cardiac function and survival and increased infarct size, heart weight, and MMP2 and MMP9 expression post-MI compared with littermates. NKRF inhibits CF migration and invasion in vitro by downregulating MMP2 and MMP9 expression. Mechanistically, NKRF inhibits human antigen R (HuR) transcription by binding to the classical negative regulatory element within the HuR promoter via an NF-κB-dependent mechanism. This decreases HuR-targeted Mmp2 and Mmp9 mRNA stability. This study suggests that NKRF is a therapeutic target for pathological cardiac remodeling.

Role of hsa_circ_0000280 in regulating vascular smooth muscle cell function and attenuating neointimal hyperplasia via ELAVL1.

The pathological proliferation of cells in vascular smooth muscle underlies neointimal hyperplasia (NIH) development during atherosclerosis. Circular RNAs (circRNAs), which represent novel functional biomarkers and RNA-binding proteins, contribute to multiple cardiovascular diseases; however, their roles in regulating the vascular smooth muscle cell cycle remain unknown. Thus, we aimed to identify the roles of circRNAs in vascular smooth muscle during coronary heart disease (CHD). Through circRNA sequencing of CHD samples and human antigen R (ELAVL1) immunoprecipitation, we identified circRNAs that are associated with CHD and interact with ELAVL1. Our results suggested that the hsa_circ_0000280 associated with CHD inhibits cell proliferation and induces ELAVL1-dependent cell cycle arrest. Gain/loss-of-function experiments and assays in vivo indicated that hsa_circ_0000280 facilitates interactions between ELAVL1 and cyclin-dependent kinase suppressor 1 (CDKN1A) mRNA and stabilization of this complex and leads to cell cycle arrest at the G1/S checkpoint, inhibiting cell proliferation of vascular smooth muscle cells in vitro and NIH in vivo. Importantly, hsa_circ_0000280 reduced neointimal thickness and smooth muscle cell proliferation in vivo. Taken together, these findings reveal a novel pathway in which hsa_circ_0000280 facilitates the regulation of ELAVL1 on CDKN1A mRNA to inhibit NIH. Therefore, measuring and modulating their expression might represent a potential diagnostic or therapeutic strategy for CHD.

An intersegmental single-cell profile reveals aortic heterogeneity and identifies a novel Malat1+ vascular smooth muscle subtype involved in abdominal aortic aneurysm formation.

The developmental origin, anatomical location, and other factors contribute to aortic heterogeneity in a physiological state. On this basis, vascular diseases occur at different ratios based on position specificity, even with the same risk factor. However, the continuous intersegmental aortic profile has been rarely reported at the single-cell level. To reveal aortic heterogeneity, we identified 15 cell subtypes from five continuous aortic segments by marker genes and functional definitions. The EC1 subtype highly expressed Vcam1 and Scarb2 genes in the aortic arch, which were reported to be associated with atherosclerosis. The newly identified Fbn1+ fibroblasts were found highly expressed in thoracic segments. More importantly, vascular smooth muscle cells (VSMCs) demonstrated a novel composition in which VSMC 4 marked with the gene Malat1 were mainly distributed in the abdominal segment. Malat1 knockout reduced MMPs and inflammatory factor production induced by Ang II in smooth muscle cells, and the Malat1 inhibitor exerted preventive, inhibitory, and reversing effects on AngII-induced abdominal aortic aneurysm (AAA) in vivo revealed by a series of animal experiments. Single-cell analysis of AngII-induced AAA tissues treated with or without the inhibitor further clarified the key role of Malat1+VSMC in the occurrence and progression of AAA. In summary, segmental gene expression and cell subtype features in normal aorta associated with different vascular diseases might provide potential therapeutic targets.

Deficient Chaperone-Mediated Autophagy Promotes Inflammation and Atherosclerosis.

RATIONALE: The NLRP3 (NLR [NOD-like receptor] family, pyrin domain containing 3) inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophages. However, whether the NLRP3 inflammasome is regulated by CMA, and the role of CMA in atherosclerosis remains unclear. OBJECTIVE: To determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis. METHODS AND RESULTS: The expression of CMA marker, LAMP-2A (lysosome-associated membrane protein type 2A), was first analyzed in ApoE-/- mouse aortas and human coronary atherosclerotic plaques, and a significant downregulation of LAMP-2A in advanced atherosclerosis in both mice and humans was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A knockout mouse strains in C57BL/6 mice and ApoE-/- mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE-/- mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL (interleukin)-1ß in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A-deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway. CONCLUSIONS: CMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1ß, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1ß-driven diseases.

Single-cell RNA sequencing analysis to characterize cells and gene expression landscapes in atrial septal defect.

This study aimed to characterize the cells and gene expression landscape in atrial septal defect (ASD). We performed single-cell RNA sequencing of cells derived from cardiac tissue of an ASD patient. Unsupervised clustering analysis was performed to identify different cell populations, followed by the investigation of the cellular crosstalk by analysing ligand-receptor interactions across cell types. Finally, differences between ASD and normal samples for all cell types were further investigated. An expression matrix of 18,411 genes in 6487 cells was obtained and used in this analysis. Five cell types, including cardiomyocytes, endothelial cells, smooth muscle cells, fibroblasts and macrophages were identified. ASD showed a decreased proportion of cardiomyocytes and an increased proportion of fibroblasts. There was more cellular crosstalk among cardiomyocytes, fibroblasts and macrophages, especially between fibroblast and macrophage. For all cell types, the majority of the DEGs were downregulated in ASD samples. For cardiomyocytes, there were 199 DEGs (42 upregulated and 157 downregulated) between ASD and normal samples. PPI analysis showed that cardiomyocyte marker gene FABP4 interacted with FOS, while FOS showed interaction with NPPA. Cell trajectory analysis showed that FABP4, FOS, and NPPA showed different expression changes along the pseudotime trajectory. Our results showed that single-cell RNA sequencing provides a powerful tool to study DEG profiles in the cell subpopulations of interest at the single-cell level. These findings enhance the understanding of the underlying mechanisms of ASD at both the cellular and molecular level and highlight potential targets for the treatment of ASD.

Hsa_circ_0030042 regulates abnormal autophagy and protects atherosclerotic plaque stability by targeting eIF4A3.

Rationale: Abnormal autophagic death of endothelial cells is detrimental to plaque structure as endothelial loss promotes lesional thrombosis. As emerging functional biomarkers, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular. This study is aimed to determine the role of hsa_circ_0030042 in abnormal endothelial cell autophagy and plaque stability. Methods: circRNA sequencing and quantitative polymerase chain reaction were performed to detect hsa_circ_0030042 expression in coronary heart disease (CHD) and human umbilical vein endothelial cells (HUVECs). Transfection of stubRFP-sensGFP-LC3 adenovirus, flow cytometry, and electron microscopy were used to identify the role of hsa_circ_0030042 in ox-LDL‒induced abnormal autophagy in vitro. Bioinformatic analysis, RNA immunoprecipitation, immunofluorescence assay and other in vitro experiments were performed to elucidate the mechanism underlying hsa_circ_0030042-mediated regulation of autophagy. To evaluate the role of hsa_circ_0030042 in atherosclerotic plaques and endothelial function, we measured the carotid artery tension and performed histopathology and immunohistochemistry analysis. Results: hsa_circ_0030042 was significantly downregulated in CHD, while upon overexpression, it acted as an endogenous eukaryotic initiation factor 4A-III (eIF4A3) sponge to inhibit ox-LDL-induced abnormal autophagy of HUVECs and maintain plaque stability in vivo. Furthermore, hsa_circ_0030042 influenced autophagy by sponging eIF4A3 and blocking its recruitment to beclin1 and forkhead box O1 (FOXO1) mRNA, while hsa_circ_0030042-induced inhibition of beclin1 and FOXO1 was counteracted by eIF4A3 overexpression or decreased hsa_circ_0030042 binding. In high-fat-diet fed ApoE-/- mice, hsa_circ_0030042 also ameliorated plaque stability and counteracted eIF4A3-induced plaque instability. Conclusions: These results demonstrate a novel pathway involving hsa_circ_0030042, eIF4A3, FOXO1, and beclin1; hence, modulating their levels may be a potential therapeutic strategy against CHD.

Circular RNA expression profiles and bioinformatic analysis in coronary heart disease.

Aim: We aimed to identify the expression profile and role of circular RNAs (circRNAs) in coronary heart disease (CHD). Materials & methods: We performed sequence analysis of circRNAs in peripheral blood mononuclear cells of 70 CHD patients and 30 controls. Eight selected circRNAs were validated using quantitative real-time polymerase chain reaction (qRT-PCR) in human atherosclerotic coronary arteries. Results: In total, 2283 downregulated and 85 upregulated circRNAs were identified in CHD. Parental genes of top 100 dysregulated-circRNAs are related to metabolism and protein modification, and 12 circRNAs might upregulate their CHD-related parental genes through miRNA sponges. Of the eight circRNAs validated in atherosclerotic coronary arteries by qRT-PCR, six were consistent with sequencing results of peripheral blood mononuclear cells. Conclusion: As potential ceRNAs, dysregulated circRNAs may be involved in CHD pathophysiology.

Attenuation of the hypoxia-induced miR-34a protects cardiomyocytes through maintenance of glucose metabolism.

Ischemic injury in the heart is associated with death of cardiomyocytes and even after decades of research there is no appropriate therapeutic intervention to treat ischemic injury. The microRNA miR-34a is known to be induced in cardiomyocytes following ischemic injury. Another hallmark of ischemic injury is impaired glycolysis. The objective of the current study was to investigate the effects of short- and long-term exposure to hypoxia on miR-34a expression on apoptosis and regulation of key glycolysis metabolic enzymes. Both repeated short-term (30 min) burst of hypoxia with intermittent reoxygenation (30 min) as well as long-term (4 h) exposure to hypoxia followed by 6 h of reoxygenation robustly induced miR-34a levels. Hypoxia induced changes in cardiac permeability and localization of the channel protein connexin 34 as well as induced apoptosis as evident by levels of cleaved-caspase 3/7 and impaired cell proliferation. Hypoxia was also associated with decreased expression of key glycolytic enzymes hexokinase-1, hexokinase-2, glucose-6-phosphate-isomerase, and pyruvate dehydrogenase kinase 1. Attenuation of hypoxia-induced miR-34a by anti-miR-34a antagomir, but not a control antagomir, decreased miR-34a levels to those observed under normoxia and also inhibited apoptosis, potentially by rescuing expression of the key glycolytic enzymes. Cumulatively, our results establish that therapeutic targeting of miR-34a via antagomir might be a potent therapeutic mechanism to treat ischemic injury in the heart.

Tofacitinib ameliorates atherosclerosis and reduces foam cell formation in apoE deficient mice.

Atherosclerosis is a chronic inflammatory cardiovascular disease with high mortality worldwide. Tofacitinib (CP-690,550), an oral small-molecule Janus kinase inhibitor, has been shown to be effective in the treatment of rheumatoid arthritis, autoimmune encephalomyelitis and ulcerative colitis. However, its protective effect against atherosclerosis remains poorly understood. The aim of the present study was to evaluate the effects of Tofacitinib on atherogenic diet (ATD)-induced atherosclerosis using apolipoprotein E deficient (apoE-/-) mice. Atherosclerosis-prone apoE-/- mice were fed with ATD and treated with or without Tofacitinib through intragastrical administration (10 mg kg-1 day-1) for 8 weeks. Our results showed that Tofacitinib did not change plasma lipids, while significantly reduced the levels of plasma pro-inflammatory cytokines IL-6 and TNF-α. It also significantly attenuated atherosclerotic plaque lesion in the aortic root and macrophages contained in plaque as shown with Mac2 immuno-staining. Peritoneal macrophages (PMC) were separated from apoE-/- mice fed with 8-week ATD, and then subjected to inflammation tests. Flow cytometry analysis of F4/80 and CD206 and mRNA levels of M1 and M2 macrophages markers showed that M1 macrophages decreased while M2 macrophages increased in Tofacitinib treated group. Expressions of other inflammatory genes also indicated an anti-inflammatory status in mice treated with Tofacitinib. Ox-LDL was used to induce foam cell formation from PMC in wild type mice, and the results displayed a reduced formation of foam cells and decreased inflammation in mice with Tofacitinib administration (1 µM). The mRNA and protein levels of ATP binding cassette subfamily A member 1 (ABCA1), a key gene involved in cholesterol efflux, remarkably increased, while it was absence of alterations in scavenger receptors expression. Therefore, we demonstrated that Tofacitinib could attenuate atherosclerosis and foam cells formation by inhibiting inflammation and upregulating ABCA1 expression.

miR-34a is a common link in both HIV- and antiretroviral therapy-induced vascular aging.

Both HIV and antiretroviral therapy could induce vascular aging with unclear mechanisms. In this study, via microarray analysis, we identified, for the first time, that miR-34a expression was significantly increased in both HIV-infected, and antiretroviral agents-treated vessels and vascular endothelial cells (ECs) from these vessels. In cultured ECs, miR-34a expression was significantly increased by HIV-Tat protein and by the antiretroviral agents, lopinavir/ritonavir. Both HIV-Tat protein and antiretroviral agents could induce EC senescence, which was inhibited by miR-34a inhibition. In contrast, EC senescence was exacerbated by miR-34a overexpression. In addition, the vascular ECs isolated from miR-34a knockout mice were resistant to HIV and antiretroviral agents-mediated senescence. In vivo, miR-34a expression in mouse vascular walls and their ECs was increased by antiretroviral therapy and by HIV-1 Tat transgenic approach. miR-34a inhibition could effectively inhibit both HIV-Tat protein and antiretroviral therapy-induced vascular aging in mice. The increased miR-34a was induced via p53, whereas Sirt1 was a downstream target gene of miR-34a in both HIV-Tat protein and antiretroviral agents-treated ECs and vessels. The study has demonstrated that miR-34a is a common link in both HIV and antiretroviral therapy-mediated vascular aging.

A Natural Model of Mouse Cardiac Myocyte Senescence.

Many cardiac aging studies are performed on mice first and then, due to difficulty in mouse cardiomyocyte culture, applied the rat neonatal cardiomyocytes to further determine the mechanisms in vitro. Now, the technological challenge of mouse cardiomyocyte culture has been overcome and there is an increasing need for the senescence models of mouse cardiomyocytes. In this study, we have demonstrated that the senescence of mouse cardiomyocytes occurred with the extended culture time as shown by the increased ß-galactosidase staining, increased p53 expression, decreased telomere activity, shorted telomere length, increased production of ROS, increased cell apoptosis, and impaired mitochondrial ΔΨm. These senescent responses shared similar results in aged mouse heart tissues in vivo. In summary, we have established and characterized a novel senescence model of mouse cardiomyocytes induced by the extended culture time in vitro. The cell model could be useful for the increased cardiac aging studies worldwide.

Patchouli alcohol attenuates experimental atherosclerosis via inhibiting macrophage infiltration and its inflammatory responses.

Patchouli alcohol (PA) is a tricyclic sesquiterpene extracted from a traditional Chinese herb pogostemonis herba. Literatures have proven that PA could inhibit inflammatory responses in various inflammatory disease models. However, whether PA could protect against atherosclerosis, a chronic vascular inflammation, is unknown. In this study, we sought to explore this issue in atherosclerosis-prone apolipoprotein E knockout mice fed an atherogenic diet, with or without daily PA intragastrical administration (40mg/kg). Our results showed that PA administration did not change plasma lipids metabolism, however, it significantly attenuated atherosclerotic plaque burdens in both the aorta and the aortic root. The lesional macrophage content, shown as Mac2 positive areas, was reduced, while the lesional smooth muscle cell and collagen content, shown as α-SMA positive areas and by Sirius red staining, respectively, was not affected in PA-treated mice, compared with non-treated controls. Aortic mRNA expression of macrophage inflammatory cytokines, including MCP-1, iNOS, IL-1ß, IL-6, CXCL9 and CXCL11, was also reduced in PA-treated mice. Therefore, we demonstrated that PA could attenuate atherosclerosis, possibly by inhibiting macrophage infiltration and its inflammatory responses.

Expression of calcitonin gene-related peptide type 1 receptor mRNA and their activity-modifying proteins in the rat nucleus accumbens.

The calcitonin receptor-like receptor (CRLR) and the orphan receptor RDC-1 have been proposed to be calcitonin gene-related peptide type 1 (CGRP1) receptors, and receptor activity-modifying proteins (RAMPs) determine the ligand specificity of CRLR. Coexpression of RAMP1 and CRLR resulted in functional CGRP1 receptors; the complex of RAMP2 or RAMP3 and CRLR created functional adrenomedullin receptor. Although high levels of CGRP binding sites in the nucleus accumbens have been reported, little is known about the expression of these novel CGRP receptors. In the present study, we used real-time quantitative RT-PCR to detect and quantitate the relative expression of CGRP, CRLR, RAMP1-3 and RDC-1 in the nucleus accumbens of intact rats and rats with inflammation. Our results demonstrate that CGRP, CRLR, RAMP1 and RAMP2 exist in the nucleus accumbens of intact rats, and that they were significantly upregulated in rats with inflammation. In contrast, no expression was detected for RDC-1 and RAMP3. These findings indicated a functional role for CGRP and its receptors in inflammation and pain modulation.