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Endothelial cell (EC) aging plays a vital role in the pathogenesis of cardiovascular disease (CVD). MicroRNAs have emerged as crucial regulators of target gene expression by inhibiting mRNA translation and/or promoting mRNA degradation. We identify an aging‐related and oxidative stress‐responsive microRNA, miR‐181b, that inhibits endothelial cell apoptosis and senescence. In gain‐ or loss‐of‐function studies, miR‐181b regulated the expression of key apoptosis markers (Bcl2, Bax, cleaved‐Caspase3) and senescence markers (p16, p21, γH2AX) and the ratio of apoptotic cells (TUNEL‐positive) and senescent cells (SA‐βgal‐positive) in H2O2‐induced ECs. Mechanistically, miR‐181b targets MAP3K3 and modulates a MAP3K3/MKK/MAPK signaling pathway. MAP3K3 knockdown recapitulated the phenotype of miR‐181b overexpression and miR‐181b was dependent on MAP3K3 for regulating EC apoptosis and senescence. In vivo, miR‐181b expression showed a negative correlation with increasing age in the mouse aorta. Endothelial‐specific deficiency of miR‐181a2b2 increased the target MAP3K3, markers of vascular senescence (p16, p21), and DNA double‐strand breaks (γH2AX) in the aorta of aged mice. Collectively, this study unveils an important role of miR‐181b in regulating vascular endothelial aging via an MAP3K3‐MAPK signaling pathway, providing new potential therapeutic targets for antiaging therapy in CVD. [ABSTRACT FROM AUTHOR]

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. 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. 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. [Display omitted] • EC-specific deletion of miR-181a2b2 accelerated atherosclerosis, cell adhesion molecule expression, and lesion macrophages. • These findings were independent of body weight, lipid metabolism, or shift in the Ly6C fractions in PBMCs or bone marrow. • RNA-Seq and scRNA-seq revealed pathways associated with EC inflammation, cell adhesion, cell cycle, and immune response. [ABSTRACT FROM AUTHOR]

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. 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. 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. [Display omitted] • We provide an optimized enzymatic digestion protocol of the aorta, ideal for scRNAseq, FACS, and cell isolation and culture. • The new magnetic beads-based selection method is rapid and efficiently isolates diverse aortic cells simultaneously. • Isolated primary aortic ECs, SMCs, leukocytes, and fibroblasts can be cultured for further vascular biology experiments. • An optimized intima peeling approach enables efficient RNA enrichment specifically from the aortic intima or media. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chronic non-healing wounds occur frequently in individuals affected by diabetes, yet standard-of-care treatment leaves many patients inadequately treated or with recurring wounds. MicroRNA (miR) expression is dysregulated in diabetic wounds and drives an anti-angiogenic phenotype, but miRs can be inhibited with short, chemically-modified RNA oligonucleotides (anti-miRs). Clinical translation of anti-miRs is hindered by delivery challenges such as rapid clearance and uptake by off-target cells, requiring repeated injections, excessively large doses, and bolus dosing mismatched to the dynamics of the wound healing process. To address these limitations, we engineered electrostatically assembled wound dressings that locally release anti-miR-92a, as miR-92a is implicated in angiogenesis and wound repair. In vitro , anti-miR-92a released from these dressings was taken up by cells and inhibited its target. An in vivo cellular biodistribution study in murine diabetic wounds revealed that endothelial cells, which play a critical role in angiogenesis, exhibit higher uptake of anti-miR eluted from coated dressings than other cell types involved in the wound healing process. In a proof-of-concept efficacy study in the same wound model, anti-miR targeting anti-angiogenic miR-92a de-repressed target genes, increased gross wound closure, and induced a sex-dependent increase in vascularization. Overall, this proof-of-concept study demonstrates a facile, translational materials approach for modulating gene expression in ulcer endothelial cells to promote angiogenesis and wound healing. Furthermore, we highlight the importance of probing cellular interactions between the drug delivery system and the target cells to drive therapeutic efficacy. [Display omitted] • Woven dressings can be conformally coated with pro-angiogenic anti-miR therapy. • Electrostatic assembly with polyelectrolytes enables packaging of anti-miR. • Anti-miR released from dressings preferentially targets wound endothelial cells. • In murine diabetic ulcers, dressings coated with anti-miR-92a improve healing. • Anti-miR-92a promotes angiogenesis in a sex-dependent manner. [ABSTRACT FROM AUTHOR]

Wound healing is a physiological reparative response to injury and a well-orchestrated process that involves hemostasis, cellular migration, proliferation, angiogenesis, extracellular matrix deposition, and wound contraction and re-epithelialization. However, patients with type 2 diabetes mellitus (T2D) are frequently afflicted with impaired wound healing that progresses into chronic wounds or diabetic ulcers, and may lead to complications including limb amputation. Herein, we investigate the potential role of microRNA-26a (miR-26a) in a diabetic model of wound healing. Expression of miR-26a is rapidly induced in response to high glucose in endothelial cells (ECs). Punch skin biopsy wounding of db/db mice revealed increased expression of miR-26a (~ 3.5-fold) four days post-wounding compared to that of WT mice. Local administration of a miR-26a inhibitor, LNA-anti-miR-26a, induced angiogenesis (up to ~ 80%), increased granulation tissue thickness (by 2.5-fold) and accelerated wound closure (53% after nine days) compared to scrambled anti-miR controls in db/db mice. These effects were independent of altered M1/M2 macrophage ratios. Mechanistically, inhibition of miR-26a increased its target gene SMAD1 in ECs nine days post-wounding of diabetic mice. In addition, high glucose reduced activity of the SMAD1-3′-UTR. Diabetic dermal wounds treated with LNA-anti-miR-26a had increased expression of ID1, a downstream modulator or SMAD1, and decreased expression of the cell cycle inhibitor p27. These findings establish miR-26a as an important regulator on the progression of skin wounds of diabetic mice by specifically regulating the angiogenic response after injury, and demonstrate that neutralization of miR-26a may serve as a novel approach for therapy. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic immune-inflammatory disorder that integrates multiple cell types and a diverse set of inflammatory mediators. miRNAs are emerging as important post-transcriptional regulators of gene expression in most, if not all, vertebrate cells, and constitute central players in many physiological and pathological processes. Rapidly accumulating experimental studies reveal their key role in cellular and molecular processes related to the development of atherosclerosis. We review current evidence for the involvement of miRNAs in early atherosclerotic lesion formation and in plaque rupture and erosion. We conclude with a perspective on the clinical relevance, therapeutic opportunities, and future challenges of miRNA biology in understanding the pathogenesis of this complex disease. [ABSTRACT FROM AUTHOR]

Emerging evidence demonstrates that proangiogenic cells (PAC5) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMP5) differentiate into PAC5 and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF β1-responsive KrUppellike factor, KLF10, is strongly expressed in PAC5 derived from CMP5 and GMPs, 60-fold higher than in progenitors lacking PAC markers. KLF10-1- mice present with marked defects in PAC differentiation, function, TGF-β1-responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10-1- PACs. Overexpression studies revealed that KLF10-1- could rescue PAC formation from TGF4-β1-1- CMPs and GMP5. Mechanistically, KLF10-1- targets the VEGFR2 promoter in PAC5 which may underlie the observed effects. These findings may be clinically relevant because KLF10-1- expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-β1-1- signaling and KLF10-1- as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. [ABSTRACT FROM AUTHOR]

CD4+CD25+ regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF1O/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4+CD25 T cell activation through distinct mechanisms involving transforming growth factor (TGF)-β1 and Foxp3. KLF10 overexpressing CD4+CD25 T cells induced both TGF-β1 and Foxp3 expression, an effect associated with reduced T-Bet (Thi marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10-/- CD4+CD25- T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10-/- CD4+CD25 T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10-/- T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-β1, an effect completely rescued by exogenous treatment with TGF-β1. Mechanistic studies demonstrate that in response to TGF-β1, KLF10 can transactivate both TGF-β1 and Foxp3 promoters, implicating KLF10 in a positive feedback ioop that may promote cell-intrinsic control of T cell activation. Finally, KLF10-/- CD4+CD25 T cells promoted atherosclerosis by ∼2-fold in ApoE-/-/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-β1 in both CD4+CD25 T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice. [ABSTRACT FROM AUTHOR]

Atherosclerotic plaque rupture mainly contributes to acute coronary syndrome (ACS). Insufficient repair of these plaques leads to thrombosis and subsequent ACS. Central to this process is the modulation of vascular smooth muscle cells (VSMCs) phenotypes, emphasizing their pivotal role in atherosclerotic plaque stability and healing post-disruption. Here, an expansion of FSP1 + cells in a tandem stenosis (TS) model of atherosclerotic mice is unveiled, predominantly originating from VSMCs through single-cell RNA sequencing (scRNA-seq) analyses and VSMC lineage tracing studies. Further investigation identified TEA domain transcription factor 1 (TEAD1) as the key transcription factor driving the trans-differentiation of VSMCs into fibroblast-like cells. In vivo experiments using a TS model of plaque rupture demonstrated that TEAD1 played a crucial role in promoting plaque stability and healing post-rupture through pharmacological or TEAD1-AAV treatments. Mechanistically, it is found that TEAD1 promoted the expression of fibroblast markers through the Wnt4/β-Catenin pathway, facilitating the trans-differentiation. Thus, this study illustrated that TEAD1 played a critical role in promoting the trans-differentiation of VSMCs into fibroblast-like cells and subsequent extracellular matrix production through the Wnt4/β-Catenin pathway. Consequently, this process enhanced the healing mechanisms following plaque rupture, elucidating potential therapeutic avenues for managing atherosclerotic instability., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Aims: Inflammation has been implicated in the pathogenesis of desmoplakin (DSP) cardiomyopathy, and retrospective studies have described abnormal myocardial fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) findings in symptomatic patients eventually diagnosed with DSP cardiomyopathy. We aimed to prospectively investigate if ambulatory patients with DSP cardiomyopathy had myocardial FDG uptake PET/CT imaging indicative of myocardial inflammation and if they had circulating biomarker evidence of inflammation., Methods: We prospectively recruited participants with DSP cardiomyopathy and participants with titin cardiomyopathy as a comparator group. Blood samples for clinical labs and proteomic profiling, myocardial perfusion single-photon emission computed tomography (SPECT) and myocardial FDG PET/CT were obtained for all participants., Results: Ten participants with DSP cardiomyopathy (median age 36.5 years (28, 60); 80% female; 100% White and non-Hispanic) and four participants with titin cardiomyopathy (median age 55.5 years [38.5, 64]; 50% female; 100% White and non-Hispanic) were recruited. There were no significant differences between the groups in white blood cell count, ESR, hsCRP or hsTn. Three participants with DSP cardiomyopathy and two participants with titin cardiomyopathy had non-specific myocardial FDG uptake on PET/CT. All other participants had no myocardial FDG uptake. Integration of miRNA differential expression and their predicted targets from the differential expression proteomics data identified a total of 11 inverse miRNA-mRNA pairs potentially involved in the regulation of top 20 significantly enriched pathways, including pathways involved in metabolism, inflammasome/inflammatory signalling and cell death/pyroptosis., Conclusions: In a group of ambulatory patients with DSP and titin cardiomyopathy, we found no differences in FDG PET/CT findings or clinical circulating biomarkers of inflammation. However, miRNA-seq/proteomics analyses identified several enriched pathways and unique miRNA-protein pairs between DSP and titin cardiomyopathy, including pathways involved in inflammasome/inflammatory signalling. Future work will centre on evaluation during myocarditis-like episodes., (© 2024 The Author(s). ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Aims: Diabetes leads to dysregulated macrophage immunometabolism, contributing to accelerated atherosclerosis progression. Identifying critical factors to restore metabolic alterations and promote resolution of inflammation remains an unmet goal. MicroRNAs orchestrate multiple signalling events in macrophages, yet their therapeutic potential in diabetes-associated atherosclerosis remains unclear., Methods and Results: miRNA profiling revealed significantly lower miR-369-3p expression in aortic intimal lesions from Ldlr-/- mice on a high-fat sucrose-containing (HFSC) diet for 12 weeks. miR-369-3p was also reduced in peripheral blood mononuclear cells from diabetic patients with coronary artery disease (CAD). Cell-type expression profiling showed miR-369-3p enrichment in aortic macrophages. In vitro, oxLDL treatment reduced miR-369-3p expression in mouse bone marrow-derived macrophages (BMDMs). Metabolic profiling in BMDMs revealed that miR-369-3p overexpression blocked the oxidized low density lipoprotein (oxLDL)-mediated increase in the cellular metabolite succinate and reduced mitochondrial respiration (OXPHOS) and inflammation [Interleukin (lL)-1β, TNF-α, and IL-6]. Mechanistically, miR-369-3p targeted the succinate receptor (GPR91) and alleviated the oxLDL-induced activation of inflammasome signalling pathways. Therapeutic administration of miR-369-3p mimics in HFSC-fed Ldlr-/- mice reduced GPR91 expression in lesional macrophages and diabetes-accelerated atherosclerosis, evident by a decrease in plaque size and pro-inflammatory Ly6Chi monocytes. RNA-Seq analyses showed more pro-resolving pathways in plaque macrophages from miR-369-3p-treated mice, consistent with an increase in macrophage efferocytosis in lesions. Finally, a GPR91 antagonist attenuated oxLDL-induced inflammation in primary monocytes from human subjects with diabetes., Conclusion: These findings establish a therapeutic role for miR-369-3p in halting diabetes-associated atherosclerosis by regulating GPR91 and macrophage succinate metabolism., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Clinical Problem: More than 200 million people worldwide have peripheral artery disease (PAD). PAD affects the quality of life and is associated with significant morbidity and mortality. Standard treatment for severe cases of PAD is surgical or endovascular revascularization. However, up to 30% of patients are not candidates for open or endovascular procedures, due to high operative risk or unfavorable vascular involvement. Furthermore, revascularization procedures may be insufficient to adequately improve microvascular tissue perfusion, wound healing, or limb salvage. Accordingly, regardless of advances in treatment modalities, outcomes of patients with PAD have remained unfavorable. Therefore, new medical therapeutic approaches are much needed. Small animal models are indispensable tools for the understanding of PAD physiopathology and the development of novel medical therapies., Recommendations for Increasing Translation From Animal Models: Development of animal models that more closely mimic the pathophysiology (with occlusive atherothrombosis and chronic development of limb ischemia) can incorporate the cardiovascular risk factors associated with this disease state, and focus on more clinically relevant outcomes is critical. In practice, this means using both animals that develop atherosclerosis and methods for the application of gradual arterial occlusion to induce hind limb ischemia. Doing so will likely help identify novel targets for intervention and overcome some principal challenges confronted by previous clinical trials. While various rodent models are discussed, the optimal animal model is yet to be defined., Competing Interests: None.

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 (R 2  = 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., Competing Interests: Disclosures None., (Copyright © 2024 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Microporous hydrogels have been widely used for delivering therapeutic cells. However, several critical issues, such as the lack of control over the harsh environment they are subjected to under pathological conditions and rapid egression of cells from the hydrogels, have produced limited therapeutic outcomes. To address these critical challenges, cell-tethering and hypoxic conditioning colloidal hydrogels containing mesenchymal stem cells (MSCs) are introduced to increase the productivity of paracrine factors locally and in a long-term manner. Cell-tethering colloidal hydrogels that are composed of tyramine-conjugated gelatin prevent cells from egressing through on-cell oxidative phenolic crosslinks while providing mechanical stimulation and interconnected microporous networks to allow for host-implant interactions. Oxygenating microparticles encapsulated in tyramine-conjugated colloidal microgels continuously generated oxygen for 2 weeks with rapid diffusion, resulting in maintaining a mild hypoxic condition while MSCs consumed oxygen under severe hypoxia. Synergistically, local retention of MSCs within the mild hypoxic-conditioned and mechanically robust colloidal hydrogels significantly increased the secretion of various angiogenic cytokines and chemokines. The oxygenating colloidal hydrogels induced anti-inflammatory responses, reduced cellular apoptosis, and promoted numerous large blood vessels in vivo. Finally, mice injected with the MSC-tethered oxygenating colloidal hydrogels significantly improved blood flow restoration and muscle regeneration in a hindlimb ischemia (HLI) model., (© 2024 Wiley‐VCH GmbH.)

Myocardial infarction (MI) is a significant cardiovascular disease that restricts blood flow, resulting in massive cell death and leading to stiff and noncontractile fibrotic scar tissue formation. Recently, sustained oxygen release in the MI area has shown regeneration ability; however, improving its therapeutic efficiency for regenerative medicine remains challenging. Here, a combinatorial strategy for cardiac repair by developing cardioprotective and oxygenating hybrid hydrogels that locally sustain the release of stromal cell-derived factor-1 alpha (SDF) and oxygen for simultaneous activation of neovascularization at the infarct area is presented. A sustained release of oxygen and SDF from injectable, mechanically robust, and tissue-adhesive silk-based hybrid hydrogels is achieved. Enhanced endothelialization under normoxia and anoxia is observed. Furthermore, there is a marked improvement in vascularization that leads to an increment in cardiomyocyte survival by ≈30% and a reduction of the fibrotic scar formation in an MI animal rodent model. Improved left ventricular systolic and diastolic functions by ≈10% and 20%, respectively, with a ≈25% higher ejection fraction on day 7 are also observed. Therefore, local delivery of therapeutic oxygenating and cardioprotective hydrogels demonstrates beneficial effects on cardiac functional recovery for reparative therapy., (© 2024 Wiley‐VCH GmbH.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Disclosures None.

Background: Previous studies demonstrate geographic and racial/ethnic variation in diagnosis and complications of diabetes and peripheral artery disease (PAD). However, recent trends for patients diagnosed with both PAD and diabetes are lacking. We assessed the period prevalence of concurrent diabetes and PAD across the United States from 2007 to 2019 and regional and racial/ethnic variation in amputations among Medicare patients., Methods: Using Medicare claims from 2007 to 2019, we identified patients with both diabetes and PAD. We calculated period prevalence of concomitant diabetes and PAD and incident cases of diabetes and PAD for every year. Patients were followed to identify amputations, and results were stratified by race/ethnicity and hospital referral region., Results: 9 410 785 patients with diabetes and PAD were identified (mean age, 72.8 [SD, 10.94] years; 58.6% women, 74.7% White, 13.2% Black, 7.3% Hispanic, 2.8% Asian/API, and 0.6% Native American). Period prevalence of diabetes and PAD was 23 per 1000 beneficiaries. We observed a 33% relative decrease in annual new diagnoses throughout the study. All racial/ethnic groups experienced a similar decline in new diagnoses. Black and Hispanic patients had on average a 50% greater rate of disease compared with White patients. One- and 5-year amputation rates remained stable at ≈1.5% and 3%, respectively. Native American, Black, and Hispanic patients were at greater risk of amputation compared with White patients at 1- and 5-year time points (5-year rate ratio range, 1.22-3.17). Across US regions, we observed differential amputation rates, with an inverse relationship between the prevalence of concomitant diabetes and PAD and overall amputation rates., Conclusions: Significant regional and racial/ethnic variation exists in the incidence of concomitant diabetes and PAD among Medicare patients. Black patients in areas with the lowest rates of PAD and diabetes are at disproportionally higher risk for amputation. Furthermore, areas with higher prevalence of PAD and diabetes have the lowest rates of amputation., Competing Interests: Disclosures None.

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-β 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-β superfamily member inhibin-β-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.

Angiogenesis is critical for tissue repair following myocardial infarction (MI), which is exacerbated under insulin resistance or diabetes. MicroRNAs are regulators of angiogenesis. We examined the metabolic regulation of miR-409-3p in post-infarct angiogenesis. miR-409-3p was increased in patients with acute coronary syndrome (ACS) and in a mouse model of acute MI. In endothelial cells (ECs), miR-409-3p was induced by palmitate, while vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) decreased its expression. Overexpression of miR-409-3p decreased EC proliferation and migration in the presence of palmitate, whereas inhibition had the opposite effects. RNA sequencing (RNA-seq) profiling in ECs identified DNAJ homolog subfamily B member 9 (DNAJB9) as a target of miR-409-3p. Overexpression of miR-409-3p decreased DNAJB9 mRNA and protein expression by 47% and 31% respectively, while enriching DNAJB9 mRNA by 1.9-fold after Argonaute2 microribonucleoprotein immunoprecipitation. These effects were mediated through p38 mitogen-activated protein kinase (MAPK). Ischemia-reperfusion (I/R) injury in EC-specific miR-409-3p knockout (KO) mice (miR-409 ECKO ) fed a high-fat, high-sucrose diet increased isolectin B4 (53.3%), CD31 (56%), and DNAJB9 (41.5%). The left ventricular ejection fraction (EF) was improved by 28%, and the infarct area was decreased by 33.8% in miR-409 ECKO compared with control mice. These findings support an important role of miR-409-3p in the angiogenic EC response to myocardial ischemia., Competing Interests: The authors have declared that no conflicts of interest exist with this work., (© 2023 The Authors.)

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., (Copyright © 2022. Published by Elsevier Inc.)

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.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Macrophages regulate inflammation and the process of tissue repair. Therefore, a better understanding of macrophages in the pathogenesis of heart failure is needed. In patients with hypertrophic cardiomyopathy, NLRC5 was significantly increased in circulating monocytes and cardiac macrophages. Myeloid-specific deletion of NLRC5 aggravated pressure overload-induced pathological cardiac remodeling and inflammation. Mechanistically, NLRC5 interacted with HSPA8 and suppressed NF-κB pathway in macrophages. The absence of NLRC5 in macrophages promoted the secretion of cytokines such as interleukin-6 (IL-6), which affected cardiomyocyte hypertrophy and cardiac fibroblast activation. Tocilizumab, an anti-IL-6 receptor antagonist, may be a novel therapeutic strategy for cardiac remodeling and chronic heart failure., Competing Interests: Funded by National Natural Science Foundation of China grants 81800424, 81900239, 81670746, 91939101, and 82070230, Clinical Research Plan of SHDC No. SHDC2020CR4019, and the Natural Science Foundation of Shanghai No. 20ZR1435300. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

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 Myh11 Cre Pad4 flox/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., (© 2022. The Author(s).)

Background Peripheral artery disease (PAD) is associated with gastrocnemius muscle abnormalities. However, the biological pathways associated with gastrocnemius muscle dysfunction and their associations with progression of PAD are largely unknown. This study characterized differential gene and microRNA (miRNA) expression in gastrocnemius biopsies from people without PAD compared with those with PAD. Participants with PAD included those with and without PAD progression. Methods and Results mRNA and miRNA sequencing were performed to identify differentially expressed genes, differentially expressed miRNAs, mRNA-miRNA interactions, and associated biological pathways for 3 sets of comparisons: (1) PAD progression (n=7) versus non-PAD (n=7); (2) PAD no progression (n=6) versus non-PAD; and (3) PAD progression versus PAD no progression. Immunohistochemistry was performed to determine gastrocnemius muscle fiber types and muscle fiber size. Differentially expressed genes and differentially expressed miRNAs were more abundant in the comparison of PAD progression versus non-PAD compared with PAD with versus without progression. Among the top significant cellular pathways in subjects with PAD progression were muscle contraction or development, transforming growth factor-beta, growth/differentiation factor, and activin signaling, inflammation, cellular senescence, and notch signaling. Subjects with PAD progression had increased frequency of smaller Type 2a gastrocnemius muscle fibers in exploratory analyses. Conclusions Humans with PAD progression exhibited greater differences in the number of gene and miRNA expression, biological pathways, and Type 2a muscle fiber size compared with those without PAD. Fewer differences were observed between people with PAD without progression and control patients without PAD. Further study is needed to confirm whether the identified transcripts may serve as potential biomarkers for diagnosis and progression of PAD.

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., (© 2022 The Authors.)

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 ( Klf10 fl/fl CD4 Cre+ ; [TKO]) and CD4-Cre ( Klf10 +/+ CD4 Cre+ ; [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.

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., (© 2022. Springer Nature Switzerland AG.)

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 IKKβ 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., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Obesity-induced insulin resistance is a risk factor for diabetes and cardiovascular disease. However, the mechanisms underlying endothelial senescence in obesity, and how it impacts obesity-induced insulin resistance remain incompletely understood. In this study, transcriptome analysis revealed that the long non-coding RNA (lncRNA) Maternally expressed gene 3 (Meg3) is one of the top differentially expressed lncRNAs in the vascular endothelium in diet-induced obese mice. Meg3 knockdown induces cellular senescence of endothelial cells characterized by increased senescence-associated β-galactosidase activity, increased levels of endogenous superoxide, impaired mitochondrial structure and function, and impaired autophagy. Moreover, Meg3 knockdown causes cellular senescence of hepatic endothelium in diet-induced obese mice. Furthermore, Meg3 expression is elevated in human nonalcoholic fatty livers and nonalcoholic steatohepatitis livers, which positively correlates with the expression of CDKN2A encoding p16, an important hallmark of cellular senescence. Meg3 knockdown potentiates obesity-induced insulin resistance and impairs glucose homeostasis. Insulin signaling is reduced by Meg3 knockdown in the liver and, to a lesser extent, in the skeletal muscle, but not in the visceral fat of obese mice. We found that the attenuation of cellular senescence of hepatic endothelium by ablating p53 expression in vascular endothelium can restore impaired glucose homeostasis and insulin signaling in obesity. In conclusion, our data demonstrate that cellular senescence of hepatic endothelium promotes obesity-induced insulin resistance, which is tightly regulated by the expression of Meg3. Our results suggest that manipulation of Meg3 expression may represent a novel approach to managing obesity-associated hepatic endothelial senescence and insulin resistance., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

CD4 + T cells regulate inflammation and metabolism in obesity. An imbalance of CD4 + T regulatory cells (Tregs) is critical in the development of insulin resistance and diabetes. Although cytokine control of this process is well understood, transcriptional regulation is not. KLF10, a member of the Kruppel-like transcription factor family, is an emerging regulator of immune cell function. We generated CD4 + -T-cell-specific KLF10 knockout (TKO) mice and identified a predisposition to obesity, insulin resistance, and fatty liver due to defects of CD4 + Treg mobilization to liver and adipose tissue depots and decreased transforming growth factor β3 (TGF-β3) release in vitro and in vivo. Adoptive transfer of wild-type CD4 + Tregs fully rescued obesity, insulin resistance, and fatty liver. Mechanistically, TKO Tregs exhibit reduced mitochondrial respiration and glycolysis, phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR signaling, and consequently impaired chemotactic properties. Collectively, our study identifies CD4 + T cell KLF10 as an essential regulator of obesity and insulin resistance by altering Treg metabolism and mobilization., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ischemic injury triggers a heightened inflammatory response that is essential for tissue repair, but excessive and chronic inflammatory responses contribute to the pathogenesis of ischemic cardiovascular disease. Regulatory T cells (Tregs), a major regulator of self-tolerance and immune suppression, control innate and adaptive immune responses, modulate specific immune cell subsets, prevent excessive inflammation, and participate in tissue repair after ischemia. Herein, we summarize the multiple potential mechanisms by which Tregs exert suppressor functions including modulation of cytokine production, alteration of cell-cell interactions, and disruption of metabolic pathways. Furthermore, we review the role of Tregs implicated in ischemic injury and repair including myocardial, limb, and cerebral ischemia. We conclude with a perspective on the therapeutic opportunities and future challenges of Treg biology in understanding the pathogenesis of ischemic cardiovascular disease states., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite major advances in the primary and secondary prevention of atherosclerosis and its risk factors, atherosclerotic cardiovascular disease remains a major clinical and financial burden on individuals and health systems worldwide. In addition, neointima formation and proliferation due to mechanical trauma to the vessel wall during percutaneous coronary interventions can lead to vascular restenosis and limit the longevity and effectiveness of coronary revascularization. Long noncoding RNAs (lncRNAs) have emerged as a novel class of epigenetic regulators with critical roles in the pathogenesis of atherosclerosis and restenosis following vascular injury. Here, we provide an in-depth review of lncRNAs that regulate the development of atherosclerosis or contribute to the pathogenesis of restenosis following mechanical vascular injury. We describe the diverse array of intracellular mechanisms by which lncRNAs exert their regulatory effects. We highlight the utility and challenges of lncRNAs as biomarkers. Finally, we discuss the immense translational potential of lncRNAs and strategies for targeting them therapeutically using oligonucleotide-based therapeutics and novel gene therapy platforms.

Peripheral artery disease, caused by chronic arterial occlusion of the lower extremities, affects over 200 million people worldwide. Peripheral artery disease can progress into critical limb ischemia (CLI), its more severe manifestation, which is associated with higher risk of limb amputation and cardiovascular death. Aiming to improve tissue perfusion, therapeutic angiogenesis held promise to improve ischemic limbs using delivery of growth factors but has not successfully translated into benefits for patients. Moreover, accumulating studies suggest that impaired downstream signaling of these growth factors (or angiogenic resistance) may significantly contribute to CLI, particularly under harsh environments, such as diabetes mellitus. Noncoding RNAs are essential regulators of gene expression that control a range of pathophysiologies relevant to CLI, including angiogenesis/arteriogenesis, hypoxia, inflammation, stem/progenitor cells, and diabetes mellitus. In this review, we summarize the role of noncoding RNAs, including microRNAs and long noncoding RNAs, as functional mediators or biomarkers in the pathophysiology of CLI. A better understanding of these ncRNAs in CLI may provide opportunities for new targets in the prevention, diagnosis, and therapeutic management of this disabling disease state.

Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.

Diabetic wound healing is an incompletely understood pathophysiological state. It comprises a range of potentially devastating and common complications of diabetes mellitus (DM) leading to intractable infections, lower extremity amputations, and associated cardiovascular morbidity and mortality. MicroRNAs (miRNAs) have emerged as important regulators in various physiological processes in health and disease through their ability to fine-tune cellular responses. Herein, we summarize the versatile roles of miRNAs implicated in diabetic wound healing in key stages including inflammation, angiogenesis, re-epithelialization, and remodeling. Furthermore, we highlight current evidence through which miRNAs exert control of gene expression and signaling pathways in the reparative response that may provide opportunities for therapeutic intervention for this potentially devastating disease state., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose of Review: Long noncoding RNAs (lncRNAs) have emerged as powerful regulators of nearly all biological processes. Their cell-type and tissue-specific expression in health and disease provides new avenues for diagnosis and therapy. This review highlights the role of lncRNAs that are involved in cardiovascular disease (CVD) with a special focus on cell types involved in cardiac injury and remodeling, vascular injury, angiogenesis, inflammation, and lipid metabolism., Recent Findings: Almost 98% of the genome does not encode for proteins. LncRNAs are among the most abundant type of RNA in the noncoding genome. Accumulating studies have uncovered novel lncRNA-mediated regulation of CVD-associated genes, signaling pathways, and pathophysiological responses. Targeting lncRNAs in vivo using short antisense oligonucleotides or by gene editing has provided important insights into disease pathogenesis through epigenetic, transcriptional, or translational mechanisms. Although cross-species conservation still remains a major obstacle, there is increasing appreciation that altered expression of lncRNAs associates with stage-specific CVD and in human patient cohorts, providing new opportunities for diagnosis and therapy., Summary: A better understanding of lncRNAs will not only fundamentally improve our understanding of key signaling pathways in CVD, but also aid in the development of effective new therapies and RNA-based biomarkers.

In this review, we focus on the emerging role of microRNAs, non-coding RNAs that regulate gene expression and signaling pathways, in dysfunctional adipose tissue. We highlight current paradigms of microRNAs involved in adipose differentiation and function in depots such as white, brown, and beige adipose tissues and potential implications of microRNA dysregulation in human disease such as obesity, inflammation, microvasculature dysfunction, and related cardiovascular diseases. We highlight accumulating studies indicating that adipocyte-derived microRNAs may not only serve as biomarkers of cardiometabolic disease, but also may directly regulate gene expression of other tissues. Finally, we discuss the future prospects, challenges, and emerging strategies for microRNA delivery and targeting for therapeutic applications in cardiovascular disease states associated with adipocyte dysfunction., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

The microRNA family, miR-181, plays diverse roles in regulating key aspects of cellular growth, development, and activation. Accumulating evidence supports a central role for the miR-181 family in vascular inflammation by controlling critical signaling pathways, such as downstream NF-κB signaling, and targets relevant to endothelial cell activation and immune cell homeostasis. This review examines the current knowledge of the miR-181 family's role in key cell types that critically control cardiovascular inflammation under pathological and physiological stimuli., (© 2013 Published by Elsevier Inc.)