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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.)

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-β 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-β secretion., Conclusions: Taken together, these findings establish a critical role for KLF10 in regulating CD4 + Treg-macrophage interactions and atherosclerosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background and Aims: Chronic vascular endothelial inflammation predisposes to atherosclerosis; however, the cell-autonomous roles for endothelial-expressing microRNAs (miRNAs) are poorly understood in this process. MiR-181b is expressed in several cellular constituents relevant to lesion formation. The aim of this study is to examine the role of genetic deficiency of the miR-181b locus in endothelial cells during atherogenesis., Methods and Results: Using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced atherosclerosis mouse model, we demonstrated that endothelial cell (EC)-specific deletion of miR-181a2b2 significantly promoted atherosclerotic lesion formation, cell adhesion molecule expression, and the influx of lesional macrophages in the vessel wall. Yet, endothelium deletion of miR-181a2b2 did not affect body weight, lipid metabolism, anti-inflammatory Ly6C low or the pro-inflammatory Ly6C interm and Ly6C high fractions in circulating peripheral blood mononuclear cells (PBMCs), and pro-inflammatory or anti-inflammatory mediators in both bone marrow (BM) and PBMCs. Mechanistically, bulk RNA-seq and gene set enrichment analysis of ECs enriched from the aortic arch intima, as well as single cell RNA-seq from atherosclerotic lesions, revealed that endothelial miR-181a2b2 serves as a critical regulatory hub in controlling endothelial inflammation, cell adhesion, cell cycle, and immune response during atherosclerosis., Conclusions: Our study establishes that deficiency of a miRNA specifically in the vascular endothelium is sufficient to profoundly impact atherogenesis. Endothelial miR-181a2b2 deficiency regulates multiple key pathways related to endothelial inflammation, cell adhesion, cell cycle, and immune response involved in the development of atherosclerosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background and Aims: Isolation of cellular constituents from the mouse aorta is commonly used for expression or functional analyses in atherosclerosis research. However, current procedures to isolate primary cells are difficult, inefficient, and require separate mice. RNA extraction from aortic intima and media for transcriptomic analysis is also considered difficult with mixed RNA yields. To address these gaps, we provide: 1) a rapid, efficient protocol to isolate and culture diverse cell types concomitantly from the mouse aorta using immunomagnetic cell isolation; and 2) an optimized aortic intimal peeling technique for efficient RNA isolation from the intima and media., Methods and Results: Aortic cells were obtained using an enzymatic solution and different cell types were isolated by magnetic beads conjugated to antibodies targeting endothelial cells (CD31 + ), leukocytes (CD45 + ), and fibroblast cells (CD90.2+), and smooth muscle cells were isolated by negative selection. Our protocol allows the isolation of relatively large numbers of cells (10,000 cells per aorta) in a predictable manner with high purity (>90%) verified by cell-marker gene expression, immunofluorescence, and flow cytometry. These cells are all functionally active when grown in cell culture. We also provide a rapid method to collect aortic intima-enriched RNA from Ldlr -/- mice utilizing an intima peeling approach and assess transcriptomic profiling associated with accelerated lesion formation., Conclusions: This protocol provides an effective means for magnetic bead-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic studies for a range of vascular diseases including atherosclerosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This Commentary highlights the article by Fang et al, which describes novel mouse models of chronic intermittent hypoxia (CIH)-induced atherosclerosis, revealing that loss of the NF-κB p50 subunit increased atherosclerosis in the presence of CIH., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Clinical studies using bone marrow-derived proangiogenic cells (PACs) have demonstrated modest improvements of function and/or perfusion of ischemic myocardium or skeletal muscle. Because the identities of these PACs and their functional ability to promote neovascularization remain poorly understood, it is possible that a subset of robust PACs exists but is obscured by the heterogeneous nature of this cell population. Herein, we found that common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) preferentially differentiate into PACs compared with megakaryocyte-erythrocyte progenitors, hematopoietic stem cells, and common lymphoid progenitors. In vivo hindlimb ischemia studies and Matrigel plug assays verified the enhanced neovascularization properties uniquely associated with PACs derived from CMPs and GMPs. Taken together, these observations identify CMPs and GMPs as key bone marrow progenitors for optimal PAC function in vitro and in vivo and provide a foundation for novel therapeutic approaches to modulate angiogenesis.

Emerging evidence demonstrates that proangiogenic cells (PACs) 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 (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-β1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ∼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-β responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-β1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-β1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.

The Krüppel-like transcription factor (KLF) family participates in diverse aspects of cellular growth, development, differentiation, and activation. Recently, several groups have identified new connections between the function of these factors and leukocyte responses in health and disease. Gene targeting of individual KLFs in mice has uncovered novel and unexpected physiologic roles among myeloid and lymphocyte cell lineage maturation, particularly in the bone marrow niche and blood. In addition, several KLF family members are downstream targets of stimuli and signaling pathways critical to T-cell trafficking, T regulatory cell differentiation or suppressor function, monocyte/macrophage activation or renewal, and B memory cell maturation or activation. Indeed, KLFs have been implicated in subtypes of leukemia, lymphoma, autoimmunity, and in acute and chronic inflammatory disease states, such as atherosclerosis, diabetes, and airway inflammation, raising the possibility that KLFs and their upstream signals are of therapeutic interest. This review focuses on the relevant literature of Krüppel-like factors in leukocyte biology and their implications in clinical settings.