Background: Peripheral artery disease is associated with significant morbidity and mortality. Mechanical revascularization strategies are a mainstay of treatment but are often limited by the anatomic complexity of atherosclerotic lesions. Therapeutic angiogenesis has fallen short of being impactful due to fundamental gaps in our understanding of postdevelopmental angiogenesis. Methods: Using a preclinical model of peripheral artery disease involving acute vascular injury by femoral artery ligation along with cellular and molecular studies of VEGF-A expression, we sought to further understand the early role of macrophages in inflammatory angiogenesis and arteriogenesis. Results: Macrophage depletion studies revealed that the optimal levels of tissue VEGF-A expression, endothelial cell recruitment, and blood flow recovery were dependent on early macrophage recruitment. Proangiogenic VEGF-A expression was highest in macrophages polarized towards an inflammatory phenotype. Myeloid VEGF-A-deletion, while having no impact on the potent inflammatory cytokine, IL-1β, led to reductions in ischemic tissue VEGF-A, endothelial cell recruitment, and blood flow recovery due to impaired angiogenesis and arteriogenesis. Transplant of inflammatory polarized macrophages rescued the myeloid VEGF-A-deletion phenotype, leading to full blood flow recovery. Conclusions: Macrophages are a necessary and sufficient source of tissue VEGF-A during inflammatory-driven angiogenesis and arteriogenesis in response to vascular injury. Although further study is needed, cell-based therapeutic angiogenesis strategies involving the polarization of macrophages toward an inflammatory state, in order to produce high levels of proangiogenic VEGF-A, may be quite effective for improving revascularization in the context of PAD.

Here, we present a protocol that models peripheral artery disease using the surgical induction of hindlimb ischemia (HLI) in mice. We list the steps to surgically excise the femoral artery and assess blood flow recovery through laser Doppler imaging. We then detail procedures for visualizing immune cell recruitment and blood vessel growth to explore the role of inflammatory macrophages in angio/arteriogenesis. For complete details on the use and execution of this protocol, please refer to Mantsounga et al.1.

Coronavirus disease 2019 (COVID-19), caused by infection with the enveloped RNA betacoronavirus, SARS-CoV-2, led to a global pandemic involving over 7 million deaths. Macrophage inflammatory responses impact COVID-19 severity; however, it is unclear whether macrophages are infected by SARS-CoV-2. We sought to identify mechanisms regulating macrophage expression of ACE2, the primary receptor for SARS-CoV-2, and to determine if macrophages are susceptible to productive infection. We developed a humanized ACE2 (hACE2) mouse whereby hACE2 cDNA was cloned into the mouse ACE2 locus under control of the native promoter. We validated the susceptibility of hACE2 mice to SARS-CoV-2 infection relative to wild-type mice and an established K18-hACE2 model of acute fulminating disease. Intranasal exposure to SARS-CoV-2 led to pulmonary consolidations with cellular infiltrate, edema, and hemorrhage, consistent with pneumonia, yet unlike the K18-hACE2 model, hACE2 mice survived and maintained stable weight. Infected hACE2 mice also exhibited a unique plasma chemokine, cytokine, and growth factor inflammatory signature relative to K18-hACE2 mice. Infected hACE2 mice demonstrated evidence of viral replication in infiltrating lung macrophages, and infection of macrophages in vitro revealed a transcriptional profile indicative of altered RNA and ribosomal processing machinery as well as activated cellular antiviral defense. Macrophage IL-1β-driven NF-κB transcription of ACE2 was an important mechanism of dynamic ACE2 upregulation, promoting macrophage susceptibility to infection. Experimental models of COVID-19 that make use of native hACE2 expression will allow for mechanistic insight into factors that can either promote host resilience or increase susceptibility to worsening severity of infection.

Diabetes mellitus (DM) is a chronic disease and a major health problem worldwide, especially in elderly people (>65 years). Patients with chronic DM suffer from vascular complications including heart failure, coronary heart disease and peripheral artery disease (PAD) . DM and aging are important risk factors of PAD. DM increases the risk of developing PAD while PAD contributes to the progression of DM . PAD-mediated DM (PAD-DM) is associated with reduced blood supply and revascularization to the lower limbs resulting in limb amputation or immobility. Current treatment options are largely limited to mechanical revascularization, by surgical bypass or angioplasty, and medical management. Critical limb ischemia in PAD-DM and wound injury animal models have shown the impact of monocytes/macrophages as a major source of angiogenic mediators like the vascular endothelial growth factor (VEGF-A) and IL-1beta (IL-1β). However, pre- and clinical investigations involving VEGF-A modestly improved revascularization, but arteriogenesis restoration was incomplete in humans and animal models. We have defined a key mechanism whereby macrophage IL-1β promotes VEGF-A expression under the regulation of transcription factors (NF-κB and STAT3) in young mice . Our data from Type 2 diabetes mellitus (TD2M) experimental mouse model, leptin receptor (Lepr db/db ) at 12-week-old demonstrated delayed blood flow recovery and new capillary growth versus young control, using a PAD model of femoral artery ligation. “Aged” 56-week-old control mice also showed reductions in blood flow recovery and angiogenesis. Moreover, combining aging with long-term diabetes, 56-week-old T2DM mice, led to further reductions in blood flow recovery consequently to impaired angiogenesis . RNA sequencing data from polarized macrophages and single nucleus RNA sequencing data from ischemic muscle tissue (gastrocnemius) from “aged” T2DM mice, showed a significant increase of Cxcl2-NLRP3 inflammasome signaling while with IL-1 receptor; MyD88 and VEGF-A expressions were reduced compared to aged control. Here, we aim to define the molecular mechanisms of altered inflammatory angiogenesis in response to vascular injury related to 1) an exacerbated and deleterious inflammation (Cxcl2-NLRP3-IL1β) and 2) a reduction in healing process (MyD88; Msi2-TGF-β signaling) in the setting of long-term diabetes mellitus. Potential results will lead to new strategies to improve neo/revascularization in PAD-DM and age-related defects.

Background and Objectives: Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairment of angiogenesis responses to injury. Critical ischemic limb and wound injury animal models have shown impact of monocytes/macrophages as a major source of angiogenic mediators leading to new arterial growth. Aging has also been associated with impairment of blood flow recovery and reduced VEGF-A expression in animals. However, recombinant VEGF-A administration or clinical trials involving VEGF-A have not fully overcome the foot perfusion and prevent limb/leg amputation. We sought to understand molecular mechanisms of reduced VEGF-A expression which remain incomplete in the context of advanced aging. Results: Firstly, in hindlimb ischemia mouse model, our data from liposomes containing-clodronate treated mice (12-weeks C57Bl6 with macrophage depletion model ) ( Fig. 1a ) demonstrate impairment of blood flow recovery compared to Control group. On day 3 post-surgery, VEGF-A and VEGF-A isoforms expression (VEGF-A 165 a and VEGF-A 165 b, respectively) were reduced in ischemic muscle. Fluorescence staining showed a reduction of macrophages (CD68 + ) was also associated with reduced endothelial cells (CD31 + ). Using a myeloid-specific and inducible deleted VEGF fl/fl mice, we then have shown a key role of early inflammatory macrophages stage as a major source of VEGF-A required for angiogenesis in young mice (Fig. 1b). Secondly, bone marrow-derived-macrophages (BMDMs) from C57Bl6 104-weeks mice also demonstrate reduced VEGF-A expression. Aged BMDMs demonstrate reduced VEGF-A 165 a while VEGF-A 165 b was highly increased. Polarized BMDMs from 12-weeks demonstrate increased VEGFR1 in “M2" macrophages while VEGFR2 was increased in “M1” macrophages. On the other hand, BMDMs from 104-weeks demonstrate reduced VEGFR2 expression while VEGFR1 was unaffected compared to young. Lastly, 104-weeks mice also demonstrate severe impairment of blood flow recovery ( Fig. 2 ) after surgery and reduced VEGF-A and VEGF-A 165 a in ischemic muscle tissue. VEGF-A 165 b was increased and both receptors, VEGF-R1/R2 levels ( Fig. 3 ) were also reduced. Fluorescence staining on day 3 post ligation demonstrates reduced endothelial cells recruitment in muscle tissue while macrophage (CD68+) number was similar. Conclusion: Our investigations will contribute to define mechanisms whereby vascular injury is associated with VEGF-A isoforms specific switch in the context of advanced aging.

Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.

Background: Chronic limb threatening ischemia (CLTI) is defined as the presence of peripheral artery disease (PAD) in combination with rest pain or tissue loss and represents advanced arterial occlusive disease. Little is known about associated venous pathology as prior pathologic reports were limited to evaluation of maximally diseased arterial lesions. Objectives: To characterize arterial and venous histopathology in patients with CLTI. Methods: Tissue containing peripheral arteries and veins from the femoropopliteal to distal tibial arteries was harvested from 10 patients who underwent amputation for CLTI (Fig 1). Vessels were sectioned at 0.5 cm intervals, and histopathologic features were catalogued across 535 arterial and 493 venous sections. Results: Consistent with PAD, atherosclerotic lesions were more prevalent in proximal than distal arteries; 81.8% (27/33) femoropopliteal sections compared to 26.6% (134/502) of tibial sections. Severe stenosis (>90%) was noted in 21.9% (117) of sections and was associated with atherosclerosis (pathological intimal thickening, fibroatheroma, fibrocalcific or fibro-osseous lesions) in 10.3% (12), luminal thrombi in 68.4% (80), or both in 21.4% (25) of arterial sections. In all patients and in 99.0% (488/493) of venous sections, irrespective of the degree of arterial luminal stenosis, adjacent large (≥1000µm) and small diameter veins revealed marked thickening of the vessel wall comprised of fibrosis and smooth muscle hyperplasia (Fig 2). While large veins retained luminal patency, small veins exhibited severe luminal stenosis or obliteration. Venous thrombosis was uncommon, occurring in 3.0% (15/493) of sections. Conclusions: Venous remodeling with stenosis and occlusion is highly prevalent and coincides with arterial disease pathology in patients with CLTI. This novel finding of previously underappreciated and severely altered venous pathology suggests a possible additional mechanism for limb ischemia in CLTI.

Background Atherosclerosis and consequent risk of cardiovascular events or mortality can be accurately assessed by quantifying coronary artery calcium score (CACS) derived from computed tomography. HMG-CoA-reductase inhibitors (statins) are the primary pharmacotherapy used to reduce cardiovascular events, yet there is growing data that support statin use may increase coronary calcification. We set out to determine the likelihood of severe CACS in the context of chronic statin therapy. Methods We established a retrospective, case-control study of 1,181 U.S. veterans without coronary artery disease (CAD) from a single site, the Providence VA Medical Center. Duration of statin therapy for primary prevention was divided into 5-year categorical increments. The primary outcome was CACS derived from low-dose lung cancer screening computed tomography (LCSCT), stratified by CACs severity (none = 0; mild = 1–99; moderate = 100–399; and severe ≥400 AU). Statin duration of zero served as the referent control. Ordinal logistic regression analysis determined the association between duration of statin use and CACS categories. Proportional odds assumption was tested using likelihood ratio test. Atherosclerotic cardiovascular disease (ASCVD) risk score, body mass index, and CKD (glomerular filtration rate of <60 ml/min/1.73 m2) were included in the adjustment models. Results The mean age of the study population was 64.7±7.2 years, and 706 (60%) patients were prescribed a statin at baseline. Duration of statin therapy was associated with greater odds of having increased CACS (>0–5 years, OR: 1.71 [CI: 1.34–2.18], p<0.001; >5–10 years, OR: 2.80 [CI: 2.01–3.90], p<0.001; >10 years, OR: 5.30 [CI: 3.23–8.70], p<0.001), and the relationship between statin duration and CACS remained significant after multivariate adjustment (>0–5 years, OR: 1.49 [CI: 1.16–1.92], p = 0.002; >5–10 years, OR: 2.38 [CI: 1.7–3.35], p<0.001; >10 years, OR: 4.48 [CI: 2.7–7.43], p<0.001). Conclusions Long-term use of statins is associated with increased likelihood of severe CACS in patients with significant smoking history. The use of CACS to interpret cardiovascular event risk may require adjustment in the context of chronic statin therapy.

HomeCirculation: Cardiovascular ImagingVol. 16, No. 3Unlocking the Secrets of Subclinical Calcific Aortic Valve Disease and Heart Failure Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBUnlocking the Secrets of Subclinical Calcific Aortic Valve Disease and Heart Failure Sheila Sharma and Alan R. Morrison Sheila SharmaSheila Sharma https://orcid.org/0000-0002-3796-2786 Department of Medicine (Section of Cardiovascular Medicine), Providence VA Medical Center, Rhode Island (S.S., A.R.M.). Department of Medicine (Section of Cardiovascular Medicine), Alpert Medical School at Brown University, Providence, Rhode Island (S.S., A.R.M.). and Alan R. MorrisonAlan R. Morrison Correspondence to: Alan R. Morrison, MD, PhD, Ocean State Research Institute, Inc. Providence VA Medical Center, Research (151), 830 Chalkstone Ave, Providence, RI 02908. Email E-mail Address: [email protected] https://orcid.org/0000-0002-2412-7669 Department of Medicine (Section of Cardiovascular Medicine), Providence VA Medical Center, Rhode Island (S.S., A.R.M.). Department of Medicine (Section of Cardiovascular Medicine), Alpert Medical School at Brown University, Providence, Rhode Island (S.S., A.R.M.). Originally published7 Mar 2023https://doi.org/10.1161/CIRCIMAGING.123.015247Circulation: Cardiovascular Imaging. 2023;16This article is a commentary on the followingAortic Valve Calcium in Relation to Subclinical Cardiac Dysfunction and Risk of Heart FailureOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 7, 2023: Ahead of Print See Article by Zhu et alIn this issue of Circulation: Cardiovascular Imaging, Zhu et al1 set out to assess the relationship between subclinical calcific aortic valve disease (CAVD) and measures of cardiac structure and function followed by the relationship between CAVD and incident heart failure (HF). The study makes use of the well-characterized Rotterdam Study cohort, developed in the 1990s with the purpose of defining etiology and natural history of various chronic diseases including CAVD.2 This study is a reminder that CAVD is not benign; yet, a complete understanding of the mechanisms underlying subclinical CAVD and cardiovascular outcomes remains just out of reach.Aortic valve sclerosis, CAVD without outflow obstruction, was once considered a relatively benign, age-related degenerative disorder. In 1997, Otto et al3 upended this notion by establishing a relationship between echocardiographic measures of aortic valve sclerosis and the clinical outcomes of death and myocardial infarction. Since that seminal study, there has been a growing body of data supporting the association between aortic valve calcification (AVC) as a marker of subclinical CAVD and outcomes. AVC derived from cardiac computed tomography was associated with physiologic measures of aortic valve stenosis (AS) by echocardiography or invasive angiography, and AVC in the range of severe AS was predictive of worse prognosis in patients.4,5 Nonzero AVC in relatively low-risk populations was predictive of worse cardiovascular outcomes in a manner partially dependent on coronary artery disease as measured by coronary artery calcification (CAC).6 Nonzero AVC also appeared predictive of all-cause mortality in a manner that was incremental to CAC.7 Additional analyses using non-ECG-gated computed tomography demonstrated increasing AVC to be associated with nonfatal myocardial infarction, nonfatal cerebrovascular accident, and mortality independent of CAC, suggesting CAVD may identify a subset of atherosclerotic phenotype not readily apparent by CAC.8 More recently, AVC has been associated with cognitive impairment in patients at high risk of cardiovascular disease, supporting a link between CAVD and cerebrovascular atherosclerotic disease.9While prior work on AVC in this area focused on cardiovascular events, the critical novel finding that Zhu et al1 identify is the association of AVC with the outcome of incident heart failure. Through a combination of careful echocardiographic measures and sensitivity analyses, the authors pry away at potential plausible mechanisms that may be at the heart of this association.Zhu and colleagues1 begin by demonstrating high (≥800) AVC scores were associated with two major structural changes, increased LV mass and increased left atrial size. The authors note that prior studies also identified the relationship between elevated AVC and LV mass, left atrial dilation, or left ventricular hypertrophy.10,11 The fact that high AVC is associated with cardiac structural changes may speak to a mechanism where AS and consequent pressure overload with demand ischemia dominate. As CAVD progresses to the point of left ventricular outflow obstruction, there is chronic elevation in the left ventricular pressure during contraction. This induces compensatory adaptive and maladaptive responses to normalize ventricular wall stress, including concentric hypertrophy and remodeling to eccentric hypertrophy, in a manner determined by sex, age, cardiovascular comorbidities, and hemodynamic severity.12–14 The authors then demonstrate a significant association between AVC and incident HF with the relationship remaining strong in the AVC ≥800 category after adjustment.1 In doing so, they draw a link between structural changes and functional consequences. Moreover, the sensitivity analysis, excluding patients with established AS, abrogated the relationship between AVC and incident HF, suggesting AS to be an important mechanism of cardiac structural change and incident HF for patients with AVC ≥800. Sensitivity analysis excluding patients with known prior coronary heart disease (CHD) or incident CHD prior to HF had no impact on the relationship between AVC ≥800 and incident HF, supporting a mechanism of AS over atherosclerosis for patients in this high AVC category.The authors appropriately assess the relationship between AVC and the competing outcome, mortality, and surprisingly find no relationship between increasing AVC and mortality. Possibilities for this lack of association relative to prior studies in the field may reflect the overall study population, which had a lower prevalence of tobacco smoking, diabetes mellitus, and coronary artery disease.1,6,8 An additional limitation, which may influence the power to assess the relationship between AVC and mortality, includes the small numbers of patients with moderate to severe AVC, AS, or CHD.Another exciting finding is that the association between the moderate AVC score category of 300 to 799 and incident HF was significant, and this relationship held after adjustment for AS, suggestive of a mechanism alternative to stenosis driving incident HF in patients with moderate AVC. Sensitivity analysis excluding patients with history of CHD did not appear to impact the association; however, excluding patients with incident CHD prior to HF did abrogate the significance of this association, suggesting that atherosclerotic disease events may be an important mechanism behind the association between moderate AVC and HF. Again, numbers are small in this category, so it is important to temper enthusiasm until larger prospective studies can verify this find. The finding that exclusion of incident CHD did impact the relationship between AVC 300 to 799 and incident HF is certainly intriguing regarding mechanism because others have postulated that CAVD may identify a phenotype of inflammatory atherosclerosis driving outcomes irrespective of severe stenosis or even traditional CAC measures of atherosclerosis.6,8 Multiple studies have demonstrated a weak association between AVC and CAC, and AVC often remains independently associated with events even when adjusted for CAC, supporting that atherosclerotic vascular disease associated with AVC may phenotypically behave differently.6–8 Perhaps, some of the strongest prior data supporting this concept of a CAVD atherosclerosis phenotype is that HMG-CoA reductase inhibitors (statins), which impact outcomes in CHD, do not appear to influence the natural progression of AS.15While the presence of AVC was associated with worse left ventricular ejection fraction, the practical clinical implications of the difference between 65.6±7% and 63.4±8% are unclear.1 Moreover, the authors acknowledge limitations that may impact insight into pathophysiology and mechanism, including lack of information regarding left ventricular function at the time of overt HF and regarding HF phenotype, HFpEF versus HFrEF.Valvular atrial fibrillation leading to thromboembolic phenomena in the microvasculature has potential to be a treatable mechanism leading to ischemia and HF. When investigators control for history of atrial fibrillation, the association between AVC and incident HF remains significant, supporting that a thromboembolic mechanism is less likely.1 Challenges adjudicating atrial fibrillation and difficulty identifying subclinical atrial fibrillation may prevent full assessment of the impact of this arrhythmia. Recent and growing data on the association between other supraventricular tachyarrhythmias and cryptogenic cerebrovascular events suggestive of thromboembolic phenomena raise the question of additional confounding that must be kept in mind.16Improved modeling of CAVD and its impact on cardiovascular disease is required to fully elucidate the mechanisms driving outcomes like HF. Mouse models remain challenging because spontaneous plaque rupture is rare.17 Moreover, few models of CAVD consistently progress to AS. The widely used apolipoprotein E-deficient mouse demonstrates evidence of CAVD but with hemodynamically significant AS in about 2% to 10% of mice.18 Coupling low-density lipoprotein receptor deficiency with apolipoprotein B-100 appears to increase the likelihood of hemodynamically significant stenosis to about 30% to 50%. The aortic valve wire injury model was developed more recently as model of AS driven by inflammatory CAVD after mechanical valve injury, but widespread use of this model has been limited due to the complicated surgical technique, which can lead to high mortality with inexperienced operators.19 The New Zealand Obese (NZO/BomHIDife) strain of mice has recently emerged as a promising model, which develops spontaneous CAVD with hemodynamically significant AS in about half of the mice at 22 weeks of age along with left and right ventricular dysfunction.20In summary, the manuscript by Zhu et al1 provides important insight into the role of subclinical CAVD driving risk of heart failure. Two leading hypotheses as to mechanism have emerged from this study with (1) a phenotype of atherosclerotic CHD being likely at moderate levels of AVC and (2) outflow obstruction and aortic stenosis with consequent ventricular remodeling being likely at high levels of AVC (Figure). Additional prospective studies are required and must include a more diverse cohort, increased representation of patients in the moderate and high categories of AVC, complete heart failure phenotyping at the time of event, extensive arrhythmia phenotyping, and multivariate adjustment that considers competing outcomes. CAVD animal modeling is evolving and may help to validate some of the underlying mechanisms that drive these associations. A thorough understanding of the mechanistic links between subclinical CAVD and heart failure will lead to identification of viable targets for therapeutic approaches designed to reduce the impact of this not-so-benign disease.Download figureDownload PowerPointFigure. Illustration of the relationship between subclinical calcific aortic valve disease (CAVD) and heart failure (HF). In this issue of Circulation: Cardiovascular Imaging, Zhu et al1 uncover an important relationship between subclinical CAVD as measured by aortic valve calcification (AVC) and HF and probe into the potential mechanisms driving this association. The primary hypotheses generated are that HF risk increases with increasing AVC and that incident coronary heart disease (CHD) may drive HF at moderate (300-799) AVC levels whereas aortic valve stenosis (AS) and adverse cardiac remodeling may drive HF at high (≥800) levels of AVC.Article InformationAcknowledgmentsDr Morrison is supported by NIH NHLBI R01HL139795, R01HL163005, and VA VHA CSR&D 1I01CX002231. The views expressed in this article are those of the authors and do not reflect the position or policy of the Department of Veterans Affairs or the US government.Disclosures None.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to: Alan R. Morrison, MD, PhD, Ocean State Research Institute, Inc. Providence VA Medical Center, Research (151), 830 Chalkstone Ave, Providence, RI 02908. Email alan_morrison@brown.eduReferences1. Zhu F, Kaiser Y, Boersma E, Bos D, Kavousi M. Aortic valve calcium in relation to subclinical cardiac dysfunction and risk of heart failure.Circ Cardiovasc Imaging. 2023; 16:e014323. doi: 10.1161/CIRCIMAGING.122.014323LinkGoogle Scholar2. Ikram MA, Brusselle G, Ghanbari M, Goedegebure A, Ikram MK, Kavousi M, Kieboom BCT, Klaver CCW, de Knegt RJ, Luik AI, et al. Objectives, design and main findings until 2020 from the Rotterdam study.Eur J Epidemiol. 2020; 35:483–517. doi: 10.1007/s10654-020-00640-5CrossrefMedlineGoogle Scholar3. Otto CM, Lind BK, Kitzman DW, Gersh BJ, Siscovick DS. Association of aortic-valve sclerosis with cardiovascular mortality and morbidity in the elderly.N Engl J Med. 1999; 341:142–147. doi: 10.1056/NEJM199907153410302CrossrefMedlineGoogle Scholar4. 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Honda S, Miyamoto T, Watanabe T, Narumi T, Kadowaki S, Honda Y, Otaki Y, Hasegawa H, Netsu S, Funayama A, et al. A novel mouse model of aortic valve stenosis induced by direct wire injury.Arterioscler Thromb Vasc Biol. 2014; 34:270–278. doi: 10.1161/ATVBAHA.113.302610LinkGoogle Scholar20. Ott C, Pappritz K, Hegemann N, John C, Jeuthe S, McAlpine CS, Iwamoto Y, Lauryn JH, Klages J, Klopfleisch R, et al. Spontaneous degenerative aortic valve disease in New Zealand obese mice.J Am Heart Assoc. 2021; 10:e023131. doi: 10.1161/JAHA.121.023131LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesAortic Valve Calcium in Relation to Subclinical Cardiac Dysfunction and Risk of Heart FailureFang Zhu, et al. Circulation: Cardiovascular Imaging. 2023;16 March 2023Vol 16, Issue 3 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/CIRCIMAGING.123.015247PMID: 36880377 Originally publishedMarch 7, 2023 KeywordsEditorialsaortic valve diseasecalcificationcomputed tomographyheart failureoutcomesPDF download Advertisement SubjectsComputerized Tomography (CT)Valvular Heart DiseaseVascular Disease

Coronary artery disease caused by atherosclerosis is a leading cause of morbidity and mortality. Vascular calcification, influenced by inflammation, is associated with atherosclerotic disease burden and increased risk of cardiovascular events. The small GTPase Rac1 is an important signal transducer of inflammatory cytokine expression. We sought to define the mechanisms whereby Rac1 and its activating guanine nucleotide exchange factor (GEF), Tiam1, regulate macrophage expression of the potent inflammatory cytokine, IL-1β, and consequent calcific atherosclerotic vascular disease. To define the role of macrophage Rac1 expression on IL-1β expression and atherosclerotic calcification, we developed mouse models on the atherosclerosis-prone background, including inducible myeloid Rac1 -deletion (mR1KO) ( CSF1 mcm Rac1 fl/fl ApoE -/-) , and global Tiam1- deletion ( Tiam1 -/- ApoE -/- ). Both Rac1-deleted and Tiam1-deleted macrophages demonstrated reduced IL-1β expression in an inflammasome-stimulation assay. Macrophage Rac1 -deletion led to decreased nuclear NF-κB activity and reduced NF-κB and GTP-Rac1 binding to the IL-1β promoter by chromatin immunoprecipitation assay (ChIP). Using an inflammatory stimulus, we found that Rac1 and NF-κB form a protein complex by Proximity Ligation Assay, which is disrupted by Rac1- deletion. Gain-of-function mutations for Rac1 rescued IL-1β expression in Rac1-deleted macrophages, and mutations in the hypervariable region of Rac1 confirmed that the nuclear localization of Rac1 is required for the rescue of IL-1β expression. This recent preliminary data demonstrated that the small GTPase, Rac1, acts as a transcriptional co-factor, helping to activate NF-κB, facilitating NF-κB’s translocation to the nucleus, and binding the IL-1β promoter. In a hyperlipidemia model of experimental atherosclerosis, macrophage Rac1- deletion reduced systemic IL-1β levels and decreased plaque calcification. Targeted inhibition of the macrophage Tiam1-Rac1 signaling complex may have potential as a therapeutic strategy to mitigate inflammatory-driven vascular calcification, given that small molecule inhibitors of Tiam1-Rac1 already exist.

The COVID-19 pandemic has resulted in over 6.7 million deaths worldwide. Patients with cardiovascular disease are at increased risk for severe illness and mortality with COVID-19. Increased systemic inflammation driven by macrophages, a key cellular source of inflammatory cytokine IL-1β, may play a role in the mechanism of SARS-CoV-2 pathogenesis. We seek to understand the mechanisms whereby inflammation associated with vascular disease may drive severity of COVID-19. Under culture conditions that mimic atherosclerotic plaque cholesterol exposure, we found that macrophage IL-1β signaling may promote expression of ACE2, the established receptor for SARS-CoV-2, through NF-κB-mediated transcription. We generated a novel, “humanized” ACE2 mouse strain ( hACE2 ) by using CRISPR-Cas9 technology to insert the human ACE2 cDNA sequence into the native mouse ACE2 locus under regulation of the native promoter. Using lung tissue, presence of hACE2 mRNA was verified by RT-qPCR and protein expression was verified by immunofluorescence staining. Overall, SARS-CoV-2 intranasal inoculation of hACE2 mice revealed milder infection when compared to the K18 promoter driven hACE2 ( K18-hACE2 ) overexpression strain, which exhibits severe infection with high morality. hACE2 mice had 100% survival after infection with stable body weight despite detectable viral burden in the lung fields. Histological analysis of the lungs revealed inflammatory infiltrate and pulmonary consolidation with alveolar hemorrhage that were comparable to the K18-hACE2 mice, and immunohistochemistry confirmed detection of viral nucleocapsid in the lung consolidations. Infected hACE2 mice also demonstrated a unique plasma cytokine signature relative to K18-hACE2 mice, including elevated levels of IL-1β, IL-1α, and VEGF-A, but decreased IL-6 and TNF-α. In summary, we have established a novel model of COVID-19 that survives infection and may facilitate comparative studies involving experimental vascular disease. The ability to model COVID-19 in the context of experimental atherosclerosis may help to define new mechanisms driving severity of SARS-CoV-2 infection.

Peripheral Artery Disease (PAD), associated with diabetes, is an atherosclerotic vascular disease with impaired angiogenesis and poor wound healing. Increasing data support early inflammation as playing a key role in setting the state for adequate recovery following acute vascular injury. Here, we sought to understand the impact of diabetes upon IL-1β-dependent angiogenesis during wound healing. We identified an important role for inflammatory macrophage IL-1β-driven vascular endothelial growth factor-A (VEGF-A) production during angiogenesis required for wound healing. To determine the role of IL-1β signaling in the context of diabetes, we used a macrophage-specific IL-1 β -deletion model ( mIL-1 β ), a macrophage-specific IL-1R -deletion model ( mIL-1R ), and a Leptin ( Lepr db/db ) model. Using a punch biopsy model, wound closure was quantified over ten days. Mice with diabetes ( Lepr db/db ) demonstrate initial wound expansion, followed by delayed wound closure. Comparable results were found using mIL-1 β mice, suggesting that mice with diabetes may phenocopy disrupted IL-1β signaling. Analysis from all three models confirmed decreased macrophage expression of pro-angiogenic VEGF-A in response to IL-1β signaling in vitro and reduced VEGF-A expression in wounds in vivo . We examined the impact of upregulating IL-1β-dependent transcription factors, STAT3 and NF-κB, to bypass the impairment conferred by diabetes and found that constitutively active STAT3 and IKK-2 (NF-κB activator) appear to reconstitute VEGF-A expression in macrophages. In summary, angiogenesis dependent healing requires the contribution of macrophage IL-1β-dependent VEGF-A expression in the early inflammatory state, and diabetes is associated with an impairment in this pathway. Therapeutic angiogenesis strategies in the context of diabetes may require upregulation of transcription factors downstream of IL-1β to restore macrophage VEGF-A expression that is required for angiogenesis-dependent wound healing.

Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairedinflammatory angiogenesis responses to injury. Critical ischemic limb and wound injury animalmodels, have shown impact of inflammatory monocytes/macrophages as a major source ofproangiogenic mediators leading to new arterial growth and healing. In hind limb ischemia mousemodel of PAD, we have recently demonstrated the loss of macrophage IL-1β expression leads todecreased VEGF-A (total) and VEGF-A165a expression (proangiogenic isoform), while VEGF-A165b (inhibitory isoform) was increased, under the regulation of transcription factors SignalTransducer and Activator of Transcription 3 (STAT3) and Nuclear Factor Kappa B (NF-κB).Previous studies on aged mice have shown impaired of blood flow recovery and reduced VEGF-A expression. Moreover, recombinant VEGF-A administration did not fully overcome the footperfusion and prevent limb salvage. The mechanisms of reduced VEGF-A expression in aged animals remains incomplete. Our data from advanced aged (104-week-old) mice demonstratereduced blood flow recovery after excision of the femoral artery and reduced VEGF-A and VEGF-A165a in ischemic muscle tissue compared to young (12-week-old) while IL-1β and VEGF-A165bwere increased. Bone marrow-derived-macrophages (BMDMs) from 104-week-old alsodemonstrate decreased VEGF-A and VEGF-A165a expression. IL-1β andVEGF-A165b were foundincreased. Lastly, polarized BMDMs from 12-week-old demonstrate increased VEGFR2expression in inflammatory “M1” macrophages while VEGFR1 was increased in alternativelyactivated “M2" macrophages. Interestingly, 104-week-old demonstrate significantly increasedVEGFR1 expression while VEGFR2 was reduced compared to 12-week-old. We hypothesize thatthe regulation of IL-1β-VEGF-A axis and the type of VEGFR1/R2 involved in the signalingcomplex determine the fate of angiogenesis responses to injury in the context of advanced age.Our goals will be to define mechanisms whereby aging is associated with uncoupling of IL-1β andVEGF-A expression then, how VEGF-A isoforms and VEGFR1/2 signaling complex axis regulateinflammatory angiogenesis.

Peripheral artery disease (PAD) leads to considerable morbidity, yet strategies for therapeutic angiogenesis fall short of being impactful. Inflammatory macrophage subsets play an important role in orchestrating post-developmental angiogenesis, but the underlying mechanisms are unclear. Here, we find that macrophage VEGF-A expression is dependent upon the potent inflammatory cytokine, IL-1β. IL-1β promotes pro-angiogenic VEGF-A165a isoform transcription via activation and promoter binding of STAT3 and NF-κB, as demonstrated by gene-deletion, gain-of-function, inhibition, and chromatin immunoprecipitation assays. Conversely, IL-1β-deletion or inhibition of STAT3 or NF-κB increases anti-angiogenic VEGF-A165b isoform expression, indicating IL-1β signaling may also direct splice variant selection. In an experimental PAD model of acute limb ischemia, macrophage IL-1β expression is required for pro-angiogenic VEGF-A expression and for VEGF-A-induced blood flow recovery via angio- or arteriogenesis. Though further study is needed, macrophage IL-1β-dependent transcription of VEGF-A via STAT3 and NF-κB may have potential to therapeutically promote angiogenesis in the setting of PAD.

Peripheral artery disease (PAD) caused by atherosclerosis leads to considerable morbidity and mortality throughout the world, in large part, due to tissue damage from both acute and chronic occlusive ischemia. Preclinical studies have identified mechanisms involving bone marrow derived macrophage (BMDM) -dependent angiogenesis in an inflammation suppressed state and we recently defined a novel IL-1beta-dependent transcriptional regulation of the pro-angiogenic isoform of VEGF-A. We sought to understand the impact of diabetes on inflammatory angiogenesis in the context of aging. We hypothesized that the uncoupling of IL-1beta and VEGF-A expression with consequent impairments in angio/arteriogenesis. Control mice at 26-week-old of age or mice with experimental diabetes have reductions of angio/arteriogenesis, using a PAD model of femoral artery ligation that involves macrophage-directed blood flow recovery. Combined aging with chronic diabetes led to further reductions in blood flow recovery consequent to impaired angiogenesis. We also found elevated VEGF-R2 (relative to VEGF-R1) expression in the inflammatory “M1” state which was associated with elevations of IL-1beta and VEGF-A. Interestingly selective inhibition of VEGF-R2 led to reduced VEGF-A expression despite stable IL-1beta levels, suggesting an uncoupling of the relationship between IL-1beta and VEGF-A. Lastly, BMDMs from aged, diabetic mice demonstrated an uncoupling of IL-1beta and VEGF-A expression and VEGF-R2 was decreased in aged, diabetic BMDMs. Defining inflammatory macrophages as key, early drivers of angio/arteriogenesis via IL-1beta and VEGF-A/VEGF-R2 signaling supports a paradigm shift away from inflammation-suppression for adequate healing, allowing for macrophage reprograming strategies that promote appropriate inflammation-dependent healing responses. Disclosure C.S.Mantsounga: None. S.Sharma: None. C.Lee: None. R.Carley: None. G.Choudhary: None. A.R.Morrison: None. Funding National Institute of Health NIGMS (P20GM103652)

It is increasingly recognized that cigarette smoke (CS) exposure increases the incidence and severity of acute respiratory distress syndrome (ARDS) in critical ill humans and animals. However, the mechanism(s) is not well understood. This study aims to investigate mechanism underlying the priming effect of CS on Pseudomonas aeruginosa-triggered acute lung injury, by using pre-clinic animal models and genetically modified mice. We demonstrated that CS impaired P. aeruginosa-induced mitophagy flux, promoted p62 accumulation, and exacerbated P. aeruginosa-triggered mitochondrial damage and NLRP3 inflammasome activation in alveolar macrophages; an effect associated with increased acute lung injury and mortality. Pharmacological inhibition of caspase-1, a component of inflammasome, attenuated CS primed P. aeruginosa-triggered acute lung injury and improved animal survival. Global or myeloid-specific knockout of IL-1β, a downstream component of inflammasome activation, also attenuated CS primed P. aeruginosa-triggered acute lung injury. Our results suggest that NLRP3 inflammasome activation is an important mechanism for CS primed P. aeruginosa-triggered acute lung injury. (total words: 155).