Association between tissue loss type and amputation risk among Medicare patients with concomitant diabetes and peripheral arterial disease.

OBJECTIVE: Prior studies have described risk factors associated with amputation in patients with concomitant diabetes and peripheral arterial disease (DM/PAD). However, the association between the severity and extent of tissue loss type and amputation risk remains less well-described. We aimed to quantify the role of different tissue loss types in amputation risk among patients with DM/PAD, in the context of demographic, preventive, and socioeconomic factors. METHODS: Applying International Classification of Diseases (ICD)-9 and ICD-10 codes to Medicare claims data (2007-2019), we identified all patients with continuous fee-for-service Medicare coverage diagnosed with DM/PAD. Eight tissue loss categories were established using ICD-9 and ICD-10 diagnosis codes, ranging from lymphadenitis (least severe) to gangrene (most severe). We created a Cox proportional hazards model to quantify associations between tissue loss type and 1- and 5-year amputation risk, adjusting for age, race/ethnicity, sex, rurality, income, comorbidities, and preventive factors. Regional variation in DM/PAD rates and risk-adjusted amputation rates was examined at the hospital referral region level. RESULTS: We identified 12,257,174 patients with DM/PAD (48% male, 76% White, 10% prior myocardial infarction, 30% chronic kidney disease). Although 2.2 million patients (18%) had some form of tissue loss, 10.0 million patients (82%) did not. The 1-year crude amputation rate (major and minor) was 6.4% in patients with tissue loss, and 0.4% in patients without tissue loss. Among patients with tissue loss, the 1-year any amputation rate varied from 0.89% for patients with lymphadenitis to 26% for patients with gangrene. The 1-year amputation risk varied from two-fold for patients with lymphadenitis (adjusted hazard ratio, 1.96; 95% confidence interval, 1.43-2.69) to 29-fold for patients with gangrene (adjusted hazard ratio, 28.7; 95% confidence interval, 28.1-29.3), compared with patients without tissue loss. No other demographic variable including age, sex, race, or region incurred a hazard ratio for 1- or 5-year amputation risk higher than the least severe tissue loss category. Results were similar across minor and major amputation, and 1- and 5-year amputation outcomes. At a regional level, higher DM/PAD rates were inversely correlated with risk-adjusted 5-year amputation rates (R2 = 0.43). CONCLUSIONS: Among 12 million patients with DM/PAD, the most significant predictor of amputation was the presence and extent of tissue loss, with an association greater in effect size than any other factor studied. Tissue loss could be used in awareness campaigns as a simple marker of high-risk patients. Patients with any type of tissue loss require expedited wound care, revascularization as appropriate, and infection management to avoid amputation. Establishing systems of care to provide these interventions in regions with high amputation rates may prove beneficial for these populations.

Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

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.

FAM222A, Part of the BET-Regulated Basal Endothelial Transcriptome, Is a Novel Determinant of Endothelial Biology and Angiogenesis-Brief Report.

BACKGROUND: BETs (bromodomain and extraterminal domain-containing epigenetic reader proteins), including BRD4 (bromodomain-containing protein 4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. METHODS: RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as FAM222A (family with sequence similarity 222 member A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using small-interference RNA silencing and lentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, matrigel plug assays, and murine neonatal oxygen-induced retinopathy. RESULTS: Resting EC RNA-Seq data indicate BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation, and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A-a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting, and Matrigel plug vascularization through coordinated modulation of VEGF (vascular endothelial growth factor) and NOTCH mediator expression in vitro, ex vivo, in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. CONCLUSIONS: BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis.

Impaired angiogenesis in diabetic critical limb ischemia is mediated by a miR-130b/INHBA signaling axis.

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.

A miRNA/CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia.

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.

High-sensitivity cardiac troponin T is associated with disease activity in patients with inflammatory arthritis.

OBJECTIVE: To investigate whether high-sensitivity cardiac troponin T (hsTnT) correlates to markers of disease activity in inflammatory arthritis (IA), and whether antirheumatic treatment influences hsTnT levels. METHODS: We assessed 115 patients with active IA (64 rheumatoid arthritis (RA), 31 psoriatic arthritis and 20 ankylosing spondylitis) before and after using methotrexate (MTX) alone or tumor necrosis factor inhibitor (TNFi) with or without MTX co-medication (TNFi±MTX). All patients starting with TNFi had been previously unsuccessfully treated with MTX monotherapy. HsTnT (measured in serum by electro-chemiluminescence immunoassay (Roche Elecsys® Troponin T- high-sensitivity)), and other clinical and laboratory parameters were evaluated at baseline, and after 6 weeks and 6 months of treatment. RESULTS: Of markers of disease activity, baseline levels of hsTnT positively correlated with Physicians' Global Assessment Score of disease activity in the total patient cohort (p = 0.039). In RA group, hsTnT positively correlated with swollen joints, Disease Activity Score for 28 joints with ESR and serum tumor necrosis factor levels (p = 0.025, p = 0.008, p = 0.01, respectively). Median hsTnT at baseline was 5.0 ng/L, and did not change significantly at 6-week visit (6.0 ng/L, p = 0.37) and 6-month visit (6.0 ng/L, p = 0.18) with either antirheumatic therapy. CONCLUSIONS: HsTnT levels were associated with inflammatory markers for IA disease activity. However, while inflammatory markers significantly improved after antirheumatic treatment, hsTnT did not change during the 6-month follow-up period.

MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia.

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.

Extracellular traps from activated vascular smooth muscle cells drive the progression of atherosclerosis.

Extracellular DNA traps (ETs) represent an immune response by which cells release essential materials like chromatin and granular proteins. Previous studies have demonstrated that the transdifferentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. This study seeks to investigate the interaction between CD68+ VSMCs and the formation of ETs and highlight its function in atherosclerosis. Here we show that ETs are inhibited, and atherosclerotic plaque formation is alleviated in male Myh11CrePad4flox/flox mice undergoing an adeno-associated-virus-8 (AAV8) mediating overexpression of proprotein convertase subtilisin/kexin type 9 mutation (PCSK9) injection and being challenged with a high-fat diet. Obvious ETs generated from CD68+ VSMCs are inhibited by Cl-amidine and DNase I in vitro. By utilizing VSMCs-lineage tracing technology and single-cell RNA sequencing (scRNA-seq), we demonstrate that the ETs from CD68+ VSMCs influence the progress of atherosclerosis by regulating the direction of VSMCs' transdifferentiation through STING-SOCS1 or TLR4 signaling pathway.

Deriving International Classification of Diseases, 9th and 10th revision, codes for identifying and following up patients with diabetic lower extremity ulcers.

Objective: Administrative claims data offer a rich data source for clinical research. However, its application to the study of diabetic lower extremity ulceration is lacking. Our objective was to create a widely applicable framework by which investigators might derive and refine the International Classification of Diseases, 9th and 10th revision (ICD-9 and ICD-10, respectively) codes for use in identifying diabetic, lower extremity ulceration. Methods: We created a seven-step process to derive and refine the ICD-9 and ICD-10 coding lists to identify diabetic lower extremity ulcers. This process begins by defining the research question and the initial identification of a list of ICD-9 and ICD-10 codes to define the exposures or outcomes of interest. These codes are then applied to claims data, and the rates of clinical events are examined for consistency with prior research and changes across the ICD-9 to ICD-10 transition. The ICD-9 and ICD-10 codes are then cross referenced with each other to further refine the lists. Results: Using this method, we started with 8 ICD-9 and 43 ICD-10 codes used to identify lower extremity ulcers in patients with known diabetes and peripheral arterial disease and examined the association of ulceration with lower extremity amputation. After refinement, we had 45 ICD-9 codes and 304 ICD-10 codes. We then grouped the codes into eight clinical exposure groups and examined the rates of amputation as a rudimentary test of validity. We found that the rate of lower extremity amputation correlated with the severity of lower extremity ulceration. Conclusions: We identified 45 ICD-9 and 304 ICD-10 ulcer codes, which identified patients at risk of amputation from diabetes and peripheral artery disease. Although further validation at the medical record level is required, these codes can be used for claims-based risk stratification for long-term outcomes assessment in the treatment of patients at risk of limb loss.

KLF10 deficiency in CD4+ T cells promotes atherosclerosis progression by altering macrophage dynamics.

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.

Long Noncoding RNAs as Therapeutic Targets.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of cellular functions including maintenance of cellular homeostasis as well as the onset and progression of disease. LncRNAs often exhibit cell-, tissue-, and disease-specific expression patterns, making them desirable therapeutic targets. LncRNAs are commonly targeted using oligonucleotide therapeutics, and advances in oligonucleotide chemistry including C2 ribose sugar modifications such as 2'-fluoro, 2'-O-methyl, and 2-O-methoxyethyl modifications; 2'4'-constrained nucleotides such as locked nucleic acids and constrained 2'-O-ethyl (cEt) nucleotides; and phosphorothioate bonds have dramatically improved efficacy of oligonucleotide therapies. Novel delivery platforms such as viral vectors and nanoparticles have also improved pharmacokinetic properties of oligonucleotides targeting lncRNAs. Accumulating pre-clinical studies have utilized these strategies to therapeutically target lncRNAs and alter progression of many different disease states including Snhg12 and Chast in cardiovascular disease, Mirt2 and HOTTIP in sepsis and autoimmune disease, and Malat1 and HOXB-AS3 in cancer. Emerging oligonucleotide conjugation methods including the use of peptide nucleic acids hold promise to facilitate targeting to specific tissue types. Here, we review recent advances in lncRNA therapeutics and provide examples of how lncRNAs have been successfully targeted in pre-clinical models of disease. Finally, we detail remaining challenges facing the lncRNA field and how advances in delivery platforms and oligonucleotide chemistry might help overcome these barriers to catalyze the translation of pre-clinical studies to successful pharmaceutical development.

Antirheumatic therapy is associated with reduced complement activation in rheumatoid arthritis.

BACKGROUND: The complement system plays an important role in pathophysiology of cardiovascular disease (CVD), and might be involved in accelerated atherogenesis in rheumatoid arthritis (RA). The role of complement activation in response to treatment, and in development of premature CVD in RA, is limited. Therefore, we examined the effects of methotrexate (MTX) and tumor necrosis factor inhibitors (TNFi) on complement activation using soluble terminal complement complex (TCC) levels in RA; and assessed associations between TCC and inflammatory and cardiovascular biomarkers. METHODS: We assessed 64 RA patients starting with MTX monotherapy (n = 34) or TNFi with or without MTX co-medication (TNFi±MTX, n = 30). ELISA was used to measure TCC in EDTA plasma. The patients were examined at baseline, after 6 weeks and 6 months of treatment. RESULTS: Median TCC was 1.10 CAU/mL, and 57 (89%) patients had TCC above the estimated upper reference limit (<0.70). Compared to baseline, TCC levels were significantly lower at 6-week visit (0.85 CAU/mL, p<0.0001), without significant differences between the two treatment regimens. Notably, sustained reduction in TCC was only achieved after 6 months on TNFi±MTX (0.80 CAU/mL, p = 0.006). Reductions in TCC after treatment were related to decreased C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and interleukin 6, and increased levels of total, high and low-density lipoprotein cholesterol. Similarly, baseline TCC was significantly related to baseline CRP, ESR and interleukin 6. Patients with endothelial dysfunction had higher baseline TCC than those without (median 1.4 versus 1.0 CAU/mL, p = 0.023). CONCLUSIONS: Patients with active RA had elevated TCC, indicating increased complement activation. TCC decreased with antirheumatic treatment already after 6 weeks. However, only treatment with TNFi±MTX led to sustained reduction in TCC during the 6-month follow-up period. RA patients with endothelial dysfunction had higher baseline TCC compared to those without, possibly reflecting involvement of complement in the atherosclerotic process in RA.

MiR-409-3p targets a MAP4K3-ZEB1-PLGF signaling axis and controls brown adipose tissue angiogenesis and insulin resistance.

Endothelial cells (ECs) within the microvasculature of brown adipose tissue (BAT) are important in regulating the plasticity of adipocytes in response to increased metabolic demand by modulating the angiogenic response. However, the mechanism of EC-adipocyte crosstalk during this process is not completely understood. We used RNA sequencing to profile microRNAs derived from BAT ECs of obese mice and identified an anti-angiogenic microRNA, miR-409-3p. MiR-409-3p overexpression inhibited EC angiogenic properties; whereas, its inhibition had the opposite effects. Mechanistic studies revealed that miR-409-3p targets ZEB1 and MAP4K3. Knockdown of ZEB1/MAP4K3 phenocopied the angiogenic effects of miR-409-3p. Adipocytes co-cultured with conditioned media from ECs deficient in miR-409-3p showed increased expression of BAT markers, UCP1 and CIDEA. We identified a pro-angiogenic growth factor, placental growth factor (PLGF), released from ECs in response to miR-409-3p inhibition. Deficiency of ZEB1 or MAP4K3 blocked the release of PLGF from ECs and PLGF stimulation of 3T3-L1 adipocytes increased UCP1 expression in a miR-409-3p dependent manner. MiR-409-3p neutralization improved BAT angiogenesis, glucose and insulin tolerance, and energy expenditure in mice with diet-induced obesity. These findings establish miR-409-3p as a critical regulator of EC-BAT crosstalk by modulating a ZEB1-MAP4K3-PLGF signaling axis, providing new insights for therapeutic intervention in obesity.

Antirheumatic therapy is not associated with changes in circulating N-terminal pro-brain natriuretic peptide levels in patients with autoimmune arthritis.

BACKGROUND: Patients with autoimmune arthritis (AA) are at increased risk for impaired cardiac function and heart failure. This may be partly due to the effect of inflammation in heart function. The impact of antirheumatic drugs on cardiac dysfunction in AA remains controversial. Therefore, we aimed to examine effects of antirheumatic treatment on serum N-terminal pro-brain natriuretic peptide (NT-proBNP) in AA patients and its relationship to inflammatory markers. METHODS: We examined 115 patients with AA (64 rheumatoid arthritis (RA), 31 psoriatic arthritis and 20 ankylosis spondylitis) starting with methotrexate (MTX) monotherapy or tumor necrosis factor inhibitors (TNFi) with or without MTX co-medication. NT-proBNP (measured in serum by ECLIA from Roche Diagnostics), and other clinical and laboratory parameters were evaluated at baseline, after 6 weeks and 6 months of treatment. RESULTS: NT-proBNP levels did not change significantly after 6 weeks and 6 months of antirheumatic therapy (pbaseline-6weeks = 0.939; pbaseline-6months = 0.485), although there was a modest improvement from 6 weeks to 6 months in the MTX only treatment group (median difference = -18.2 [95% CI = -32.3 to -4.06], p = 0.013). There was no difference in the effects of MTX monotherapy and TNFi regimen on NT-proBNP levels. The changes in NT-proBNP after antirheumatic treatment positively correlated with changes in C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Baseline NT-proBNP levels were related to baseline CRP and ESR levels, and some other established markers of disease activities in crude analyses. CONCLUSION: Circulating levels of NT-proBNP were related to established inflammatory markers at baseline, and the changes in NT-proBNP after antirheumatic treatment were positively related to these markers. Nevertheless, antirheumatic therapy did not seem to affect NT-proBNP levels compared to baseline, even though inflammatory markers significantly improved.

The subcellular redistribution of NLRC5 promotes angiogenesis via interacting with STAT3 in endothelial cells.

Angiogenesis is a critical step in repair of tissue injury. The pattern recognition receptors (PRRs) recognize pathogen and damage associated molecular patterns (DAMPs) during injury and achieve host defense directly. However, the role of NLR family CARD domain containing 5 (NLRC5), an important member of PPRs, beyond host defense in angiogenesis during tissue repair remains unknown. Methods:In vitro, western blot and real-time PCR (RT-PCR) were used to detect the expression of NLRC5 in endothelial cells (ECs). Immunofluorescence microscopy was used to reveal the subcellular location of NLRC5 in ECs. Cell proliferation, wound healing, tube formation assays of ECs were performed to study the role of NLRC5 in angiogenesis. By using Tie2Cre-NLRC5flox/flox mice and bone marrow transplantation studies, we defined an EC-specific role for NLRC5 in angiogenesis. Mechanistically, co-immunoprecipitation studies and RNA sequencing indicated that signal transducer and activator of transcription 3 (STAT3) was the target of NLRC5 in the nucleus. And Co-IP was used to verify the specific domain of NLRC5 binding with STAT3. ChIP assay determined the genes regulated by interaction of STAT3 and NLRC5. Results: Knockdown of NLRC5 in vitro or in vivo inhibited pathological angiogenesis, but had no effect on physiological angiogenesis. NLRC5 was also identified to bind to STAT3 in the nucleus required the integrated death-domain and nucleotide-binding domain (DD+NACHT domain) of NLRC5. And the interaction of STAT3 and NLRC5 could enhance the transcription of angiopoietin-2 (Ang2) and cyclin D1 (CCND1) to participate in angiogenesis. Conclusions: In the ischemic microenvironment, NLRC5 protein accumulates in the nucleus of ECs and enhances STAT3 transcriptional activity for angiogenesis. These findings establish NLRC5 as a novel modulator of VEGFA signaling, providing a new target for angiogenic therapy to foster tissue regeneration.

MicroRNA-17-3p suppresses NF-κB-mediated endothelial inflammation by targeting NIK and IKKß binding protein.

Nuclear factor kappa B (NF-κB) activation contributes to many vascular inflammatory diseases. The present study tested the hypothesis that microRNA-17-3p (miR-17-3p) suppresses the pro-inflammatory responses via NF-κB signaling in vascular endothelium. Human umbilical vein endothelial cells (HUVECs), transfected with or without miR-17-3p agomir/antagomir, were exposed to lipopolysaccharide (LPS), and the inflammatory responses were determined. The cellular target of miR-17-3p was examined with dual-luciferase reporter assay. Mice were treated with miR-17-3p agomir and the degree of LPS-induced inflammation was determined. In HUVECs, LPS caused upregulation of miR-17-3p. Overexpression of miR-17-3p in HUVECs inhibited NIK and IKKß binding protein (NIBP) protein expression and suppressed LPS-induced phosphorylation of inhibitor of kappa Bα (IκBα) and NF-κB-p65. The reduced NF-κB activity was paralleled by decreased protein levels of NF-κB-target gene products including pro-inflammatory cytokine [interleukin 6], chemokines [interleukin 8 and monocyte chemoattractant protein-1] and adhesion molecules [vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin]. Immunostaining revealed that overexpression of miR-17-3p reduced monocyte adhesion to LPS-stimulated endothelial cells. Inhibition of miR-17-3p with antagomir has the opposite effect on LPS-induced inflammatory responses in HUVECs. The anti-inflammatory effect of miR-17-3p was mimicked by NIBP knockdown. In mice treated with LPS, miR-17-3p expression was significantly increased. Systemic administration of miR-17-3p for 3 days suppressed LPS-induced NF-κB activation and monocyte adhesion to endothelium in lung tissues of the mice. In conclusion, miR-17-3p inhibits LPS-induced NF-κB activation in HUVECs by targeting NIBP. The findings therefore suggest that miR-17-3p is a potential therapeutic target/agent in the management of vascular inflammatory diseases.

Methotrexate attenuates vascular inflammation through an adenosine-microRNA-dependent pathway.

Endothelial cell (EC) activation is an early hallmark in the pathogenesis of chronic vascular diseases. MicroRNA-181b (Mir181b) is an important anti-inflammatory mediator in the vascular endothelium affecting endotoxemia, atherosclerosis, and insulin resistance. Herein, we identify that the drug methotrexate (MTX) and its downstream metabolite adenosine exert anti-inflammatory effects in the vascular endothelium by targeting and activating Mir181b expression. Both systemic and endothelial-specific Mir181a2b2-deficient mice develop vascular inflammation, white adipose tissue (WAT) inflammation, and insulin resistance in a diet-induced obesity model. Moreover, MTX attenuated diet-induced WAT inflammation, insulin resistance, and EC activation in a Mir181a2b2-dependent manner. Mechanistically, MTX attenuated cytokine-induced EC activation through a unique adenosine-adenosine receptor A3-SMAD3/4-Mir181b signaling cascade. These findings establish an essential role of endothelial Mir181b in controlling vascular inflammation and that restoring Mir181b in ECs by high-dose MTX or adenosine signaling may provide a potential therapeutic opportunity for anti-inflammatory therapy.

A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus.

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR-/- mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.

LncRNA VINAS regulates atherosclerosis by modulating NF-κB and MAPK signaling.

Long noncoding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNA-Seq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INflammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDL receptor-deficient (LDLR-/-) mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1ß, and COX-2, in endothelial cells (ECs), vascular smooth muscle cells, and bone marrow-derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEP domain containing 4 (DEPDC4), an evolutionary conserved human ortholog of VINAS with approximately 74% homology, showed similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated antiinflammatory effects of VINAS in human ECs. These findings reveal a potentially novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.