Adenosine kinase inhibition protects mice from abdominal aortic aneurysm via epigenetic modulation of VSMC inflammation.

AIM: Abdominal aortic aneurysm (AAA) is a common, serious vascular disease with no effective pharmacological treatment. The nucleoside adenosine plays an important role in modulating vascular homeostasis, which prompted us to determine whether adenosine kinase (ADK), an adenosine metabolizing enzyme, modulates AAA formation via control of intracellular adenosine level, and to investigate the underlying mechanisms. METHODS AND RESULTS: We used a combination of genetic and pharmacological approaches in murine models of AAA induced by calcium chloride (CaCl2) application or angiotensin II (Ang II) infusion to study the role of ADK in the development of AAA. In vitro functional assays were performed by knocking down ADK with adenovirus-short hairpin RNA in human vascular smooth muscle cells (VSMCs), and the molecular mechanisms underlying ADK function were investigated using RNA-sequencing, isotope tracing and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR). Heterozygous deficiency of Adk protected mice from CaCl2- and Ang II-induced AAA formation. Moreover, specific knockout of Adk in VSMCs prevented Ang II-induced AAA formation, as evidenced by reduced aortic extracellular elastin fragmentation, neovascularization and aortic inflammation. Mechanistically, ADK knockdown in VSMCs markedly suppressed the expression of inflammatory genes associated with AAA formation, and these effects were independent of adenosine receptors. Metabolic flux and ChIP-qPCR results showed that ADK knockdown in VSMCs decreased S-adenosylmethionine (SAM)-dependent transmethylation, thereby reducing H3K4me3 binding to the promoter regions of the genes that are associated with inflammation, angiogenesis and extracellular elastin fragmentation. Furthermore, the ADK inhibitor ABT702 protected mice from CaCl2-induced aortic inflammation, extracellular elastin fragmentation and AAA formation. CONCLUSION: Our findings reveal a novel role for ADK inhibition in attenuating AAA via epigenetic modulation of key inflammatory genes linked to AAA pathogenesis.

AGPAT3 Deficiency Impairs Adipocyte Differentiation and Leads to a Lean Phenotype in Mice.

Acylglycerophosphate acyltransferases (AGPATs) catalyze the de novo formation of phosphatidic acid to synthesize glycerophospholipids and triglycerides. AGPATs demonstrate unique physiological roles despite a similar biochemical function. AGPAT3 is highly expressed in the testis, kidney, and liver, with intermediate expression in adipose tissue. Loss of Agpat3 is associated with reproductive abnormalities and visual dysfunction. However, the role of AGPAT3 in adipose tissue and whole-body metabolism has not been investigated. We found that male Agpat3-KO mice exhibited reduced body weights with decreased white and brown adipose tissue mass. Such changes were less pronounced in the female Agpat3-KO mice. Agpat3-KO mice have reduced plasma insulin growth factor 1 (IGF1) and insulin levels and diminished circulating lipid metabolites. They manifested intact glucose homeostasis and insulin sensitivity despite a lean phenotype. Agpat3-KO mice maintained an energy balance with normal food intake, energy expenditure, and physical activity, except for increased water intake. Their adaptive thermogenesis was also normal despite reduced brown adipose mass and triglyceride content. Mechanistically, Agpat3 was elevated during mouse and human adipogenesis and enriched in adipocytes. Agpat3-knockdown 3T3-L1 cells and Agpat3-deficient mouse embryonic fibroblasts (MEFs) have impaired adipogenesis in vitro. Interestingly, pioglitazone treatment rescued the adipogenic deficiency in Agpat3 deficient cells. We conclude that AGPAT3 regulates adipogenesis and adipose development. It is possible that adipogenic impairment in Agpat3 deficient cells potentially leads to reduced adipose mass. Findings from this work support the unique role of AGPAT3 in adipose tissue.

Transgenic Overexpression of HDAC9 Promotes Adipocyte Hypertrophy, Insulin Resistance and Hepatic Steatosis in Aging Mice.

Histone deacetylase (HDAC) 9 is a negative regulator of adipogenic differentiation, which is required for maintenance of healthy adipose tissues. We reported that HDAC9 expression is upregulated in adipose tissues during obesity, in conjunction with impaired adipogenic differentiation, adipocyte hypertrophy, insulin resistance, and hepatic steatosis, all of which were alleviated by global genetic deletion of Hdac9. Here, we developed a novel transgenic (TG) mouse model to test whether overexpression of Hdac9 is sufficient to induce adipocyte hypertrophy, insulin resistance, and hepatic steatosis in the absence of obesity. HDAC9 TG mice gained less body weight than wild-type (WT) mice when fed a standard laboratory diet for up to 40 weeks, which was attributed to reduced fat mass (primarily inguinal adipose tissue). There was no difference in insulin sensitivity or glucose tolerance in 18-week-old WT and HDAC9 TG mice; however, at 40 weeks of age, HDAC9 TG mice exhibited impaired insulin sensitivity and glucose intolerance. Tissue histology demonstrated adipocyte hypertrophy, along with reduced numbers of mature adipocytes and stromovascular cells, in the HDAC9 TG mouse adipose tissue. Moreover, increased lipids were detected in the livers of aging HDAC9 TG mice, as evaluated by oil red O staining. In conclusion, the experimental aging HDAC9 TG mice developed adipocyte hypertrophy, insulin resistance, and hepatic steatosis, independent of obesity. This novel mouse model may be useful in the investigation of the impact of Hdac9 overexpression associated with metabolic and aging-related diseases.

PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

Examining the interplay between cardiovascular disease and cancer incidence: Data from NHANES III and continuous.

Introduction: This study aimed to investigate the relationship between risk factors of cancer among individuals with existing cardiovascular disease (CVD). Methods: The analysis included 438 and 2100 CVD patients aged 65+ from NHANES-III and Continuous datasets, respectively. Competing risk models with subdistribution hazards ratio (aHR) were used to identify risk factors. Results: Females in NHANES-III had lower cancer risk (aHR 0.39, P = 0.001) compared to males. Poor physical activity was associated with increased cancer risk in both datasets (aHR 2.59 in NHANES-III, aHR 1.59 in Continuous). In NHANES-Continuous, age (aHR 1.07, P < 0.001) and current smoking (aHR 2.55, P = 0.001) also showed a significant association with developing cancer. No other factors investigated showed significant associations. Discussion: This study highlights the interplay between traditional risk factors and the elevated risk of cancer in CVD patients. Further research with larger samples and wider age ranges is needed to solidify these findings and inform intervention strategies.

Potential Therapeutic Targets for Hypotension in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is marked by genetic mutations occurring in the DMD gene, which is widely expressed in the cardiovascular system. In addition to developing cardiomyopathy, patients with DMD have been reported to be susceptible to the development of symptomatic hypotension, although the mechanisms are unclear. Analysis of single-cell RNA sequencing data has identified potassium voltage-gated channel subfamily Q member 5 (KCNQ5) and possibly ryanodine receptor 2 (RyR2) as potential candidate hypotension genes whose expression is significantly upregulated in the vascular smooth muscle cells of DMD mutant mice. We hypothesize that heightened KCNQ5 and RyR2 expression contributes to decreased arterial blood pressure in patients with DMD. Exploring pharmacological approaches to inhibit the KCNQ5 and RyR2 channels holds promise in managing the systemic hypotension observed in individuals with DMD. This avenue of investigation presents new prospects for improving clinical outcomes for these patients.

Blockade of endothelial adenosine receptor 2 A suppresses atherosclerosis in vivo through inhibiting CREB-ALK5-mediated endothelial to mesenchymal transition.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2 A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe-/- mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe-/- mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFß-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo , deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

Risk factors and mortality in dialysis patients with abdominal aortic aneurysm: A retrospective cohort study.

In the general population, abdominal aortic aneurysm (AAA) is synonymous with vascular disease and associated with increased mortality. Vascular disease is common in end-stage renal disease (ESRD) patients on dialysis, but there is limited information on AAA in this population. To address this issue, we queried the United States Renal Data System for risk factors associated with a diagnosis of AAA as well as the impact of AAA on ESRD patient survival. Incident dialysis patients from 2005 to 2014 with AAA and other clinical comorbidities were identified using ICD-9 and ICD-10 codes. Time to death was defined using the time from the start of dialysis to the date of death or to December 31, 2015. Cox proportional hazards (CPH) modeling was used to determine the adjusted hazard ratio (aHR) and 95% confidence intervals (CI) for death. From a total cohort of 820,826, we identified 21,631 subjects with a diagnosis of AAA. When compared to patients without AAA, AAA patients were older and more likely to be of white race and male gender, have a higher mean Charlson comorbidity index (CCI), have hypertension as the ESRD etiology, and use tobacco. Although a bivariate CPH model showed that AAA patients had an increased mortality risk compared to patients without the diagnosis, in the final CPH model, AAA patients had a decreased risk of mortality (aHR = 0.83, 95% CI 0.81-0.84) due to confounding with age. These results suggest that AAA is not associated with increased risk of death in ESRD patients after controlling for various demographic and clinical risk factors.

Impact of housing temperature on adipose tissue HDAC9 expression and adipogenic differentiation in high fat-fed mice.

OBJECTIVE: Impaired adipogenic differentiation exacerbates metabolic disease in obesity. This study reported that high-fat diet (HFD)-fed mice housed at thermoneutrality exhibited impaired adipogenic differentiation, attributed to increased expression of histone deacetylase 9 (HDAC9). However, the impact of HFD on adipogenic differentiation is reportedly variable, possibly reflecting divergent environmental conditions such as housing temperature. METHODS: C57BL/6J (wild-type [WT]) mice were housed at either thermoneutral (28-30°C) or ambient (20-22°C) temperature and fed HFD or chow diet (CD) for 12 weeks. For acute exposure experiments, WT or transient receptor potential cation channel subfamily M member 8 (TRPM8) knockout mice housed under thermoneutrality were acutely exposed to ambient temperature for 6 to 24 h. RESULTS: WT mice fed HFD and housed at thermoneutrality, compared with ambient temperature, gained more weight despite reduced food intake. They likewise exhibited increased inguinal adipose tissue HDAC9 expression and reduced adipogenic differentiation in vitro and in vivo compared with CD-fed mice. Conversely, HFD-fed mice housed at ambient temperature exhibited minimal change in adipose HDAC9 expression or adipogenic differentiation. Acute exposure of WT mice to ambient temperature reduced adipose HDAC9 expression independent of sympathetic ß-adrenergic signaling via a TRPM8-dependent mechanism. CONCLUSIONS: Adipose HDAC9 expression is temperature sensitive, regulating adipogenic differentiation in HFD-fed mice housed under thermoneutrality.

Integrative multiomics analysis of neointima proliferation in human saphenous vein: implications for bypass graft disease.

Introduction: Human saphenous veins (SV) are widely used as grafts in coronary artery bypass (CABG) surgery but often fail due to neointima proliferation (NP). NP involves complex interplay between vascular smooth muscle cells (VSMC) and fibroblasts. Little is known, however, regarding the transcriptomic and proteomic dynamics of NP. Here, we performed multi-omics analysis in an ex vivo tissue culture model of NP in human SV procured for CABG surgery. Methods and results: Histological examination demonstrated significant elastin degradation and NP (indicated by increased neointima area and neointima/media ratio) in SV subjected to tissue culture. Analysis of data from 73 patients suggest that the process of SV adaptation and NP may differ according to sex and body mass index. RNA sequencing confirmed upregulation of pro-inflammatory and proliferation-related genes during NP and identified novel processes, including increased cellular stress and DNA damage responses, which may reflect tissue trauma associated with SV harvesting. Proteomic analysis identified upregulated extracellular matrix-related and coagulation/thrombosis proteins and downregulated metabolic proteins. Spatial transcriptomics detected transdifferentiating VSMC in the intima on the day of harvesting and highlighted dynamic alterations in fibroblast and VSMC phenotype and behavior during NP. Specifically, we identified new cell subpopulations contributing to NP, including SPP1 + , LGALS3 + VSMC and MMP2 + , MMP14 + fibroblasts. Conclusion: Dynamic alterations of gene and protein expression occur during NP in human SV. Identification of the human-specific molecular and cellular mechanisms may provide novel insight into SV bypass graft disease.

A Lost Bullet in the Coronary Sinus: A Cautionary Tale.

We present a case of venous bullet embolism to the right atrium following a gunshot wound (GSW) to the abdomen. A 53-year-old male presented after a GSW to the abdomen. His workup included a computed tomography (CT) scan demonstrating an aortic injury with aortocaval fistula. A radio-opaque object consistent with a bullet was visualized in the right atrium. First, this case details an important decision, choice of surgery versus an interventional approach. After repair of the aortocaval fistula, the patient underwent a planned attempt to extract the bullet through a right lateral thoracotomy approach utilizing cardiopulmonary bypass to facilitate a right atriotomy. Intraoperatively, the team was not able to localize the bullet in the right atrium despite fluoroscopic evaluation. A postoperative CT scan demonstrated that the bullet had migrated into the coronary sinus. Lastly, this case demonstrates successful positioning maneuvers to dislodge the bullet out of the heart and into the inferior vena cava, allowing for the endovascular extraction of the bullet.

Extracellular Vesicles for Muscle Atrophy Treatment.

Skeletal muscle atrophy is a progressive chronic disease associated with various conditions, such as aging, cancer, and muscular dystrophy. Interleukin-6 (IL-6) is highly correlated with or plays a crucial role in inducing skeletal muscle atrophy. Extracellular vehicles (EVs), including exosomes, mediate cell-cell communication, and alterations in the genetic material contained in EVs during muscle atrophy may impair muscle cell signaling. Transplantation of muscle progenitor cell-derived EVs (MPC-EVs) is a promising approach for treating muscle diseases such as Duchenne muscular dystrophy (DMD). Moreover, stem cell-derived EVs with modification of microRNAs (e.g., miR-26 and miR-29) have been reported to attenuate muscle atrophy. Unbiased RNA-Seq analysis suggests that MPC-EVs may exert an inhibitory effect on IL-6 pathway. Here, we review the latest advances concerning the mechanisms of stem cell/progenitor cell-derived EVs in alleviating muscle atrophy, including anti-inflammatory and anti-fibrotic effects. We also discuss the clinical application of EVs in the treatment of muscle atrophy.

Ufmylation of UFBP1 Is Dispensable for Endoplasmic Reticulum Stress Response, Embryonic Development, and Cardiac and Intestinal Homeostasis.

Protein modification by ubiquitin fold modifier 1 (UFM1), termed ufmylation, regulates various physiological and pathological processes. Among emerging UFM1 targets, UFM1 binding protein 1 (UFBP1) is the first identified ufmylation substrate. Recent clinical and animal studies have demonstrated the pivotal roles of UFBP1 in development, hematopoiesis, intestinal homeostasis, chondrogenesis, and neuronal development, which has been linked to its function in maintaining endoplasmic reticulum (ER) homeostasis. However, the importance of UFBP1 ufmylation in these cellular and physiological processes has yet to be determined. It has been proposed that ufmylation of lysine 268 (267 in humans) in UFBP1 plays a critical role in mediating the effects of the ufmylation pathway. In this study, we for the first time probe the pathophysiological significance of UFBP1 ufmylation in vivo by creating and characterizing a mouse UFBP1 knockin (KI) model in which the lysine 268 of UFBP1, the amino acid accepting UFM1, was mutated to arginine. Our results showed that the K268R mutation reduced the total ufmylated proteins without altering the expression levels of individual ufmylation enzymes in mouse embryonic fibroblasts. The K268R mutation did not alter ER stress-stimuli-induced ER stress signaling or cell death in mouse embryonic fibroblasts. The homozygous KI mice were viable and morphologically indistinguishable from their littermate wild-type controls up to one year of age. Serial echocardiography revealed no cardiac functional impairment of the homozygous KI mice. Furthermore, the homozygous KI mice exhibited the same susceptibility to dextran sulfate sodium (DSS) -induced colitis as wild-type mice. Taken together, these results suggest that UFBP1 K268 is dispensable for ER stress response, embryonic development, cardiac homeostasis under physiological conditions, and intestinal homeostasis under pathological conditions. Our studies call for future investigations to understand the biological function of UFBP1 ufmylation and offer a new mouse model to determine the roles of UFBP1 ufmylation in different tissues under stress conditions.

Hepatocyte-specific disruption of soluble epoxide hydrolase attenuates abdominal aortic aneurysm formation: novel role of the liver in aneurysm pathogenesis.

Introduction: Inflammation is a key pathogenic feature of abdominal aortic aneurysm (AAA). Soluble epoxide hydrolase (sEH) is a pro-inflammatory enzyme that converts cytochrome P450-derived epoxides of fatty acids to the corresponding diols, and pharmacological inhibition of sEH prevented AAA formation. Both cytochrome P450 enzymes and sEH are highly expressed in the liver. Here, we investigated the role of hepatic sEH in AAA using a selective pharmacological inhibitor of sEH and hepatocyte-specific Ephx2 (which encodes sEH gene) knockout (KO) mice in two models of AAA [angiotensin II (AngII) infusion and calcium chloride (CaCl 2 ) application]. Methods and results: sEH expression and activity were strikingly higher in mouse liver compared with aorta and further increased the context of AAA, in conjunction with elevated expression of the transcription factor Sp1 and the epigenetic regulator Jarid1b, which have been reported to positively regulate sEH expression. Pharmacological sEH inhibition, or liver-specific sEH disruption, achieved by crossing sEH floxed mice with albumin-cre mice, prevented AAA formation in both models, concomitant with reduced expression of hepatic sEH as well as complement factor 3 (C3) and serum amyloid A (SAA), liver-derived factors linked to AAA formation. Moreover, sEH antagonism markedly reduced C3 and SAA protein accumulation in the aortic wall. Co-incubation of liver ex vivo with aneurysm-prone aorta resulted in induction of sEH in the liver, concomitant with upregulation of Sp1, Jarid1b, C3 and SAA gene expression, suggesting that the aneurysm-prone aorta secretes factors that activate sEH and downstream inflammatory signaling in the liver. Using an unbiased proteomic approach, we identified a number of dysregulated proteins [ e.g., plastin-2, galectin-3 (gal-3), cathepsin S] released by aneurysm-prone aorta as potential candidate mediators of hepatic sEH induction. Conclusion: We provide the first direct evidence of the liver's role in orchestrating AAA via the enzyme sEH. These findings not only provide novel insight into AAA pathogenesis, but they have potentially important implications with regard to developing effective medical therapies for AAA.

Identification of the metabolic state of surviving cardiomyocytes in the human infarcted heart by spatial single-cell transcriptomics.

The metabolic status of surviving cardiomyocytes (CM) in the myocardial tissues of patients who sustained myocardial infarction (MI) is largely unknown. Spatial single-cell RNA-sequencing (scRNA-seq) is a novel tool that enables the unbiased analysis of RNA signatures within intact tissues. We employed this tool to assess the metabolic profiles of surviving CM in the myocardial tissues of patients post-MI. Methods: A spatial scRNA-seq dataset was used to compare the genetic profiles of CM from patients with MI and control patients; we analyzed the metabolic adaptations of surviving CM within the ischemic niche. A standard pipeline in Seurat was used for data analysis, including normalization, feature selection, and identification of highly variable genes using principal component analysis (PCA). Harmony was used to remove batch effects and integrate the CM samples based on annotations. Uniform manifold approximation and projection (UMAP) was used for dimensional reduction. The Seurat "FindMarkers" function was used to identify differentially expressed genes (DEGs), which were analyzed by the Gene Ontology (GO) enrichment pathway. Finally, the scMetabolism R tool pipeline with parameters method = VISION (Vision is a flexible system that utilizes a high-throughput pipeline and an interactive web-based report to annotate and explore scRNA-seq datasets in a dynamic manner) and metabolism.type = Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to quantify the metabolic activity of each CM. Results: Analysis of spatial scRNA-seq data showed fewer surviving CM in infarcted hearts than in control hearts. GO analysis revealed repressed pathways in oxidative phosphorylation, cardiac cell development, and activated pathways in response to stimuli and macromolecular metabolic processes. Metabolic analysis showed downregulated energy and amino acid pathways and increased purine, pyrimidine, and one-carbon pool by folate pathways in surviving CM. Conclusions: Surviving CM within the infarcted myocardium exhibited metabolic adaptations, as evidenced by the downregulation of most pathways linked to oxidative phosphorylation, glucose, fatty acid, and amino acid metabolism. In contrast, pathways linked to purine and pyrimidine metabolism, fatty acid biosynthesis, and one-carbon metabolism were upregulated in surviving CM. These novel findings have implications for the development of effective strategies to improve the survival of hibernating CM within the infarcted heart.

INKILN is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation via Scaffolding MKL1 and USP10.

BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1ß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.

Perivascular adipose tissue promotes vascular dysfunction in murine lupus.

Introduction: Patients with systemic lupus erythematosus (SLE) are at elevated risk for Q10 cardiovascular disease (CVD) due to accelerated atherosclerosis. Compared to heathy control subjects, lupus patients have higher volumes and densities of thoracic aortic perivascular adipose tissue (PVAT), which independently associates with vascular calcification, a marker of subclinical atherosclerosis. However, the biological and functional role of PVAT in SLE has not been directly investigated. Methods: Using mouse models of lupus, we studied the phenotype and function of PVAT, and the mechanisms linking PVAT and vascular dysfunction in lupus disease. Results and discussion: Lupus mice were hypermetabolic and exhibited partial lipodystrophy, with sparing of thoracic aortic PVAT. Using wire myography, we found that mice with active lupus exhibited impaired endothelium-dependent relaxation of thoracic aorta, which was further exacerbated in the presence of thoracic aortic PVAT. Interestingly, PVAT from lupus mice exhibited phenotypic switching, as evidenced by "whitening" and hypertrophy of perivascular adipocytes along with immune cell infiltration, in association with adventitial hyperplasia. In addition, expression of UCP1, a brown/beige adipose marker, was dramatically decreased, while CD45-positive leukocyte infiltration was increased, in PVAT from lupus mice. Furthermore, PVAT from lupus mice exhibited a marked decrease in adipogenic gene expression, concomitant with increased pro-inflammatory adipocytokine and leukocyte marker expression. Taken together, these results suggest that dysfunctional, inflamed PVAT may contribute to vascular disease in lupus.

Electrical Stimulation Increases the Secretion of Cardioprotective Extracellular Vesicles from Cardiac Mesenchymal Stem Cells.

Clinical trials have shown that electric stimulation (ELSM) using either cardiac resynchronization therapy (CRT) or cardiac contractility modulation (CCM) approaches is an effective treatment for patients with moderate to severe heart failure, but the mechanisms are incompletely understood. Extracellular vesicles (EV) produced by cardiac mesenchymal stem cells (C-MSC) have been reported to be cardioprotective through cell-to-cell communication. In this study, we investigated the effects of ELSM stimulation on EV secretion from C-MSCs (C-MSCELSM). We observed enhanced EV-dependent cardioprotection conferred by conditioned medium (CM) from C-MSCELSM compared to that from non-stimulated control C-MSC (C-MSCCtrl). To investigate the mechanisms of ELSM-stimulated EV secretion, we examined the protein levels of neutral sphingomyelinase 2 (nSMase2), a key enzyme of the endosomal sorting complex required for EV biosynthesis. We detected a time-dependent increase in nSMase2 protein levels in C-MSCELSM compared to C-MSCCtrl. Knockdown of nSMase2 in C-MSC by siRNA significantly reduced EV secretion in C-MSCELSM and attenuated the cardioprotective effect of CM from C-MSCELSM in HL-1 cells. Taken together, our results suggest that ELSM-mediated increases in EV secretion from C-MSC enhance the cardioprotective effects of C-MSC through an EV-dependent mechanism involving nSMase2.