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Diabetic kidney disease (DKD) is the most significant complication in diabetic patients, ultimately leading to renal fibrosis. The most important manifestation of DKD is the epithelial-mesenchymal transition (EMT) of renal tubular cells, which can lead to renal fibrosis and inflammatory injury in special situations. Sphingosine 1-phosphate (S1P) is involved in various signal transduction pathways and plays a role through G protein-coupled receptors. Research has demonstrated that blocking the S1P / S1PR2 pathway inhibits inflammation and fibrosis. However, the interaction between S1P/S1PR1 and the pathophysiology of EMT remains ambiguous. The purpose of this study was to investigate the mechanism of S1P/S1PR1 on high glucose (HG)-induced renal EMT. We found that HG markedly increased the S1P and EMT marker levels in renal tubular epithelial cells. At the same time, HG could stimulate NF-κB/ROS/NLRP3 expression, but these phenomena were reversed after blocking S1PR1. In mice models of DKD, FTY720 (S1P antagonist) could significantly improve renal function and reduce the infiltration of inflammatory cells. ROS, as well as NLPR3 inflammasome, were markedly decreased in the treatment group. FTY720 inhibits extracellular matrix synthesis and improves renal fibrosis. In brief, the HG stimulates S1P/S1PR1 synthesis and activates the S1P/S1PR1 pathway. Through the S1P/S1PR1 pathway, activates NF-κB, promotes ROS generation and NLRP3 inflammasome activation, and ultimately causes EMT.

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Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

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To predict massive cerebral infarction (MCI) occurrence after anterior circulation occlusion (ACO) by cASPECTS-CTA-CS (combined ASPECTS and CTA-CS). Of 185 cerebral infarction patients with the ACO, their collateral circulation scores from CT angiography (CTA) images in two groups (MCI and non-MCI) were evaluated using Alberta Stroke Program Early CT Score (ASPECTS) and CT angiography collateral score (CTA-CS) approaches. The cASPECTS-CTA-CS was validated internally using the bootstrap sampling method with 1000 bootstrap repetitions and compared to CTA-CS. Receiver-operating characteristic curve (ROC), clinical impact curve (CIC), and decision curve analysis (DCA) strategies were used to assess the clinical practicality and predictability of both approaches (cASPECTS-CTA-CS and CTA-CS). Using net reclassification improvement (NRI) and integrated discrimination improvement (IDI) analyses, discrimination levels of the cASPECTS-CTA-CS were compared with CTA-CS. Classification and regression tree (CART) analyses was conducted to identify the best predictive values and identify subgroup of MCI. The discrimination ability of collateral circulation evaluation score using the cASPECTS-CTA-CS [AUC: 0.918, 95% confidence interval (CI): 0.869-0.967, P < 0.01; NRI: 0.200, 95% CI: -0.104 to 0.505, P = 0.197; and IDI: 0.107, 95% CI: 0.035-0.178, P = 0.004] was better than CTA-CS alone (AUC: 0.885, 95% CI: 0.833-0.937, P < 0.01). DCA indicated the net benefits of the cASPECTS-CTA-CS approach was higher than CTA-CS alone when the threshold probability range over 20%. CIC analyses showed that the number of high risks and true positives were in agreement when the threshold probability > 80%. Less than 23 of cASPECTS-CTA-CS by CART was important factor in determining MCI occurrence, and ASPECTS < 7 was followed factor. The cASPECTS-CTA-CS approach cumulatively predicted MCI after ACO.

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BACKGROUND AND PURPOSE: We examined functional outcomes of mechanical thrombectomy (MT) procedures following anterior circulation large vessel occlusion (ACLVO)-related acute ischemic strokes (AIS). Results were based on admission non-contrast computed tomography (NCCT) studies, using the Alberta Stroke Program Early Computed Tomography Score (ASPECTS) as standard metric. METHODS: Qualifying subjects were consecutive patients (N = 343) at a single center undergoing MT for ACLVO-related AIS. Each was grouped according to ASPECTS status on admission, determined from NCCT images by two physicians. Primary clinical endpoint was functional independence, assessed via modified Rankin Scale (mRS) at 90 days. Secondary endpoints were vessel recanalization (i.e., modified Thrombolysis in Cerebral Infarction [mTICI] score), symptomatic intracranial hemorrhage (sICH), and mortality. RESULTS: In this study population (mean age, 63.6 ± 12.6 years; women, 30.3%; median baseline National Institute of Health Stroke Scale [NIHSS] score, 15.2 ± 4.5), patients were stratified by ASPECTS tier at presentation, either 0-5 (n = 50) or 6-10 (n = 293). Multivariate logistic regression showed a relation between ASPECTS values ≤ 5 and lesser chance of 90-day functional improvement (OR = 2.309, 95% confidence interval [CI] 1.012-5.271; p = 0.047), once adjusted for age, baseline NIHSS score, diabetes mellitus, HbA1c concentration, D-dimer level, occlusive location, numbers of device passes, and successful recanalization. CONCLUSIONS: ASPECTS values ≤ 5 correspond with worse long-term functional improvement (mRS scores > 2) in patients undergoing MT for ACLVO-related AIS. Other independent determinants of functional outcomes after MT are age, baseline NIHSS score, HbA1c concentration, and successful recanalization.

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Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) as well as the leading cause of mortality in patients. Previous studies revealed that S1P level is elevated in plasma samples of SLE patients and murine lupus models. FTY720, targeting S1P receptors, exhibited therapeutic effects in improving the nephritis symptoms of lupus mouse models. However, few studies have discussed the potential relevance of S1P/S1PR to the pathogenesis of LN. Macrophages have been shown to be an important causative agent of renal inflammation, while the pro-inflammatory M1-type promotes kidney injury and inflammation during LN. Importantly, macrophages express various S1P receptors, and how they respond to S1P in the setting of LN remains unclear. Therefore, we examined the level of S1P in the lupus MRL/lpr mice and explored the ensuing interaction of macrophages and S1P. We found that S1P level was elevated in the MRL/lpr mice with a subsequent enhancement of the S1PR1 expression, and blocking S1PR1 by FTY720, the nephritis symptoms of MRL/lpr mice were improved. Mechanistically, we demonstrated that elevated S1P level increase the M1-type macrophage accumulation. And the in-vitro studies proved that S1P/S1PR1 was involved in the promotion of macrophage polarization towards M1 type through activation of NLRP3 inflammasome. These findings confer a novel role to macrophage S1PR1 and provide a new perspective for targeting S1P during LN.

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Systemic lupus erythematosus (SLE) is a chronic multisystem inflammatory disease associated with autoantibody formation. Lupus nephritis (LN) is one of the most severe organ manifestations of SLE. The inflammatory response is a key factor in kidney injury, and the NLRP3 inflammasome is frequently associated with the pathogenesis of LN. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD +)-dependent histone deacetylase, is a promising therapeutic target for preventing renal injury. However, the mechanism of SIRT1 in LN remains unclear. Here, we aimed to investigate the mechanism by which SIRT inhibits the NLRP3 inflammasome to slow the progression of LN. We detected the expression of SIRT1 and the infiltration of macrophages in MRL/lpr mice; the results showed that the expression of SIRT1 was decreased, and the symptoms of lupus nephritis were relieved after the use of resveratrol, which upregulated SIRT1. In vitro studies showed that after lipopolysaccharide (LPS) stimulation, SIRT1 expression decreased, and the NLRP3 inflammasome was activated. Upregulation of SIRT1 inhibits NLRP3 inflammasome activation and assembly by interfering with two signalling pathways. First, SIRT1 affects NF-κB expression, transcription, and inflammatory cytokine expression. Second, SIRT1 modulates calcium influx induced by transient receptor potential channel M2 (TRPM2), which could be partly due to the inhibition of reactive oxygen species (ROS) production. Our findings suggest that upregulated SIRT1 inhibits the NLRP3 inflammasome to slow the progression of lupus nephritis by regulating NF-κB and ROS/TRPM2/Ca2+ channels. This study reveals a new anti-inflammatory mechanism of SIRT1, suggesting that SIRT1 may be a potential therapeutic target for the prevention of LN.

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Patients with peripheral artery disease (PAD) and diabetes compose a high-risk population for development of critical limb ischemia (CLI) and amputation, although the underlying mechanisms remain poorly understood. Comparison of dysregulated microRNAs from diabetic patients with PAD and diabetic mice with limb ischemia revealed the conserved microRNA, miR-130b-3p. In vitro angiogenic assays demonstrated that miR-130b rapidly promoted proliferation, migration, and sprouting in endothelial cells (ECs), whereas miR-130b inhibition exerted antiangiogenic effects. Local delivery of miR-130b mimics into ischemic muscles of diabetic mice (db/db) following femoral artery ligation (FAL) promoted revascularization by increasing angiogenesis and markedly improved limb necrosis and amputation. RNA-Seq and gene set enrichment analysis from miR-130b-overexpressing ECs revealed the BMP/TGF-ß signaling pathway as one of the top dysregulated pathways. Accordingly, overlapping downregulated transcripts from RNA-Seq and miRNA prediction algorithms identified that miR-130b directly targeted and repressed the TGF-ß superfamily member inhibin-ß-A (INHBA). miR-130b overexpression or siRNA-mediated knockdown of INHBA induced IL-8 expression, a potent angiogenic chemokine. Lastly, ectopic delivery of silencer RNAs (siRNA) targeting Inhba in db/db ischemic muscles following FAL improved revascularization and limb necrosis, recapitulating the phenotype of miR-130b delivery. Taken together, a miR-130b/INHBA signaling axis may provide therapeutic targets for patients with PAD and diabetes at risk of developing CLI.

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Rapamycin has great potential in the antitumor application, but its therapeutic effect is seriously affected by poor water solubility, targeting ability, and low bioavailability. Here, we constructed a novel composite nanomaterial with PCN-224 as a drug carrier and loaded rapamycin, named R@BP@HA. The nanoplate not only improves targeting, but also synergizes rapamycin with PCN-224 to effectively promote tumor cell apoptosis, which subsequently causes immunogenic cell death (ICD), and shows strong therapeutic effect in 4T1 breast cancer model. The treatment effect depends on three main points:(i)Proapoptotic effect of rapamycin on tumor cells;(ii)ROS production by PCN-224-mediated photodynamic therapy;(iii)ICD induced DC maturation, increased immune response and promoted T cell proliferation and differentiation. This nanoplate offers potential antitumor efficacy in combination with chemotherapy, photodynamic therapy, and immunotherapy.

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Patients with peripheral artery disease (PAD) and diabetes have the highest risk of critical limb ischemia (CLI) and amputation, yet the underlying mechanisms remain incompletely understood. MicroRNA (miRNA) sequencing of plasma from diabetic patients with or without CLI was compared to diabetic mice with acute or subacute limb ischemia to identify conserved miRNAs. miRNA-KO mice on high-fat diet were generated to explore the impact on CLI. Comparison of dysregulated miRNAs from diabetic individuals with PAD and diabetic mice with limb ischemia revealed conserved miR-181 family members. High-fat-fed, diabetic Mir181a2b2-KO mice had impaired revascularization in limbs due to abrogation of circulating Ly6Chi monocytes, with reduced accumulation in ischemic skeletal muscles. M2-like KO macrophages under diabetic conditions failed to produce proangiogenic cytokines. Single-cell transcriptomics of the bone marrow niche revealed that the reduced monocytosis in diabetic KO mice was a result of impaired hematopoiesis, with increased CXCR4 signaling in bone marrow Lineage-Sca1+Kit+ (LSK) cells. Exogenous Ly6Chi monocytes from nondiabetic KO mice rescued the impaired revascularization in ischemic limbs of diabetic KO mice. Increased Cxcr4 expression was mediated by the miR-181 target, Plac8. Taken together, our results show that MiR-181a/b is a putative mediator of diabetic CLI and contributes to changes in hematopoiesis, monocytosis, and macrophage polarization.

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After massive afforestation, the Loess Plateau is facing the severe challenge of water shortages. Water use efficiency (WUE) is an important indicator of plant drought resistance, and high WUE is an important way to reconcile the contradiction between vegetation growth and soil water consumption (SWC). Different vegetation types significantly influence hydrological cycle process and WUE. In this study, the Biome-BGC model was used to simulate and analyze the soil water storage (SWS), SWC, and WUE of 3 typical vegetation types in the Loess Plateau from 2005 to 2020. The results showed that the order of SWS of different vegetation types from largest to smallest was grassland (GL, 81.82 mm/day), abandoned farmland (AF, 66.92 mm/day), and Robinia pseudoacacia forest (RP, 55.64 mm/day); SWC was RP (480.09 mm/year), GL (464.68 mm/year), and AF (421.79 mm/year); WUE was RP (2.37 gC/kgH2O), GL (1.10 gC/kgH2O), and AF (0.60 gC/kgH2O). GL showed a better water retention capacity. Precipitation recharge did not meet the full SWC of vegetation. In years of high vegetation growth, as well as in the dry season when water was scarce, both RP and GL showed varying degrees of water deficit. Correlation analysis revealed that a positive effect of precipitation on WUE has a threshold effect, and the thresholds range from approximately 15-50 mm/day for RP, 15-25 mm/day for GL, and no clear pattern for AF. Overall, in water-stressed areas, a large expansion of forest land should be reduced and GL should be increased. In seasons and areas where vegetation is growing vigorously or extremely arid, irrigation regarding precipitation thresholds should be carried out to improve the WUE of vegetation and promote the sustainable development of regional ecology.

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Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.

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Diabetes mellitus is a global public health problem whose cases will continue to rise along with the progressive increase in obesity and the aging of the population. People with diabetes exhibit higher risk of cardiovascular complications, especially myocardial infarction (MI). microRNAs (miRNAs) are evolutionary conserved small non-coding RNAs involved in the regulation of biological processes by interfering in gene expression at the post-transcriptional level. Accumulating studies in the last two decades have uncovered the role of stage-specific miRNAs associated with key pathobiological events observed in the hearts of people with diabetes and MI, including cardiomyocyte death, angiogenesis, inflammatory response, myocardial remodeling, and myocardial lipotoxicity. A better understanding of the importance of these miRNAs and their targets may provide novel opportunities for RNA-based therapeutic interventions to address the increased risk of MI in diabetes.

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Objective: The purpose of this study is to establish and evaluate an early biomarker prediction model of massive cerebral infarction caused by anterior circulation occlusion. Methods: One hundred thirty-four patients with acute cerebral infarction from January 2018 to October 2020 were selected to establish the development cohort for the internal test of the nomogram. Ninety-one patients with acute cerebral infarction hospitalized in our hospital from December 2020 to December 2021 were constituted the validation cohort for the external validation. All patients underwent baseline computed tomography (CT) scans within 12 h of onset and early imaging signs (hyperdense middle cerebral artery sign, obscuration of the lentiform nucleus, insular ribbon sign) of acute cerebral infarction were identified on CT by two neurologists. Based on follow-up CT images, patients were then divided into a massive cerebral infarction group and a non-massive cerebral infarction group. The nomogram model was constructed based on logistic regression analysis with R language. The nomogram was subsequently validated in an independent external validation cohort. Accuracy and discrimination of the prediction model were evaluated by a calibration chart, receiver operating characteristic (ROC) curve, and decision curve. Results: The indicators, including insular ribbon sign, reperfusion therapy, National Institutes of Health Stroke Scale (NHISS) score, previous cerebral infarction, and atrial fibrillation, were entered into the prediction model through binary logistic regression analysis. The prediction model showed good predictive ability. The area under the ROC curve of the prediction model was 0.848. The specificity, sensitivity, and Youden index were 0.864, 0.733, and 0.597, respectively. This nomogram to the validation cohort also showed good discrimination (AUC = 0.940, 95% CI 0.894-0.985) and calibration. Conclusion: Demonstrating favorable predictive efficacy and reproducibility, this study successfully established a prediction model of CT imaging signs and clinical data as early biomarkers of massive cerebral infarction caused by anterior circulation occlusion.

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BACKGROUND AND AIMS: Accumulating evidence supports a critical role for CD4+ T cells as drivers and modifiers of the chronic inflammatory response in atherosclerosis. Effector T cells have pro-atherogenic properties, whereas CD4+ regulatory T cells (Tregs) exert suppressive activity in atherosclerosis through increased secretion of inhibitory cytokines such as transforming growth factor-ß or interleukin-10. In addition, Tregs have been shown to suppress inflammatory macrophages and promote the resolution of atherosclerosis plaques. Impaired Treg numbers and function have been associated with atherosclerosis plaque development. However, the underlying mechanisms remain unclear. METHODS AND RESULTS: Here, we investigated a cell-autonomous role of a transcription factor, Krüppel-like factor 10 (KLF10), in CD4+ T cells in regulating atherosclerosis progression. Using CD4+ T-cell-specific KLF10 knockout (TKO) mice, we identified exaggerated plaque progression due to defects in immunosuppressive functions of Tregs on macrophages. TKO mice exhibited increased lesion size as well as higher CD4+ T cells and macrophage content compared to WT mice. TKO plaques also showed increased necrotic cores along with defective macrophage efferocytosis. In contrast, adoptive cellular therapy using WT Tregs abrogated the accelerated lesion progression and deleterious effects in TKO mice. Intriguingly, RNA-seq analyses of TKO lesions revealed increased chemotaxis and cell proliferation, and reduced phagocytosis compared to WT lesions. Mechanistically, TKO-Tregs impaired the efferocytosis capacity of macrophages in vitro and promoted a pro-inflammatory macrophage phenotype via increased IFN-γ and decreased TGF-ß secretion. CONCLUSIONS: Taken together, these findings establish a critical role for KLF10 in regulating CD4+ Treg-macrophage interactions and atherosclerosis.

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The metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is a long noncoding RNA (lncRNA) and is known for its role in cancer development and prognosis. In this study, we report that MALAT1 plays an important role in regulating acute inflammatory responses in sepsis. In patient samples, MALAT1 expression was positively correlated with severity of sepsis. In cultured macrophages, LPS treatment significantly induced MALAT1 expression, while genetic ablation of MALAT1 greatly reduced proinflammatory cytokine levels. Furthermore, MALAT1-ablated mice had significantly increased survival rates in cecal ligation and puncture (CLP)-induced sepsis and LPS-induced endotoxemia. One novel and salient feature of MALAT1-ablated mice is greatly reduced ROS level in macrophages and other cell types and increased glutathione/oxidized glutathione (GSH/GSSG) ratio in macrophages, suggesting an increased antioxidant capacity. We showed a mechanism for MALAT1 ablation leading to enhanced antioxidant capacity is through activation of methionine cycle by epitranscriptomical regulation of methionine adenosyltransferase 2A (MAT2A). MAT2A 3'UTR can be methylated by METTL16 which was known to directly bind to MALAT1. MALAT1 ablation was found to reduce methylation in MAT2A hairpin1 and increase MAT2A protein levels. Our results suggest a MALAT1-METTL16-MAT2A interactive axis which may be targeted for treatments of sepsis.

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BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

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BACKGROUND AND AIMS: Chronic vascular endothelial inflammation predisposes to atherosclerosis; however, the cell-autonomous roles for endothelial-expressing microRNAs (miRNAs) are poorly understood in this process. MiR-181b is expressed in several cellular constituents relevant to lesion formation. The aim of this study is to examine the role of genetic deficiency of the miR-181b locus in endothelial cells during atherogenesis. METHODS AND RESULTS: Using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced atherosclerosis mouse model, we demonstrated that endothelial cell (EC)-specific deletion of miR-181a2b2 significantly promoted atherosclerotic lesion formation, cell adhesion molecule expression, and the influx of lesional macrophages in the vessel wall. Yet, endothelium deletion of miR-181a2b2 did not affect body weight, lipid metabolism, anti-inflammatory Ly6Clow or the pro-inflammatory Ly6Cinterm and Ly6Chigh fractions in circulating peripheral blood mononuclear cells (PBMCs), and pro-inflammatory or anti-inflammatory mediators in both bone marrow (BM) and PBMCs. Mechanistically, bulk RNA-seq and gene set enrichment analysis of ECs enriched from the aortic arch intima, as well as single cell RNA-seq from atherosclerotic lesions, revealed that endothelial miR-181a2b2 serves as a critical regulatory hub in controlling endothelial inflammation, cell adhesion, cell cycle, and immune response during atherosclerosis. CONCLUSIONS: Our study establishes that deficiency of a miRNA specifically in the vascular endothelium is sufficient to profoundly impact atherogenesis. Endothelial miR-181a2b2 deficiency regulates multiple key pathways related to endothelial inflammation, cell adhesion, cell cycle, and immune response involved in the development of atherosclerosis.

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BACKGROUND AND AIMS: Isolation of cellular constituents from the mouse aorta is commonly used for expression or functional analyses in atherosclerosis research. However, current procedures to isolate primary cells are difficult, inefficient, and require separate mice. RNA extraction from aortic intima and media for transcriptomic analysis is also considered difficult with mixed RNA yields. To address these gaps, we provide: 1) a rapid, efficient protocol to isolate and culture diverse cell types concomitantly from the mouse aorta using immunomagnetic cell isolation; and 2) an optimized aortic intimal peeling technique for efficient RNA isolation from the intima and media. METHODS AND RESULTS: Aortic cells were obtained using an enzymatic solution and different cell types were isolated by magnetic beads conjugated to antibodies targeting endothelial cells (CD31+), leukocytes (CD45+), and fibroblast cells (CD90.2+), and smooth muscle cells were isolated by negative selection. Our protocol allows the isolation of relatively large numbers of cells (10,000 cells per aorta) in a predictable manner with high purity (>90%) verified by cell-marker gene expression, immunofluorescence, and flow cytometry. These cells are all functionally active when grown in cell culture. We also provide a rapid method to collect aortic intima-enriched RNA from Ldlr-/- mice utilizing an intima peeling approach and assess transcriptomic profiling associated with accelerated lesion formation. CONCLUSIONS: This protocol provides an effective means for magnetic bead-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic studies for a range of vascular diseases including atherosclerosis.

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Cellular reprogramming through targeting microRNAs (miRNAs) holds promise for regenerative therapy due to their profound regulatory effects in proliferation, differentiation, and function. We hypothesized that transdifferentiation of vascular smooth muscle cells (SMCs) into endothelial cells (ECs) using a miRNA cassette may provide a novel approach for use in vascular disease states associated with endothelial injury or dysfunction. miRNA profiling of SMCs and ECs and iterative combinatorial miRNA transfections of human coronary SMCs revealed a 4-miRNA cassette consisting of miR-143-3p and miR-145-5p inhibitors and miR-146a-5p and miR-181b-5p mimics that efficiently produced induced endothelial cells (iECs). Transcriptome profiling, protein expression, and functional studies demonstrated that iECs exhibit high similarity to ECs. Injected iECs restored blood flow recovery even faster than conventional ECs in a murine hindlimb ischemia model. This study demonstrates that a 4-miRNA cassette is sufficient to reprogram SMCs into ECs and shows promise as a novel regenerative strategy for endothelial repair.

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