Macrophage Homing Research Articles

A sequential macrophage activation strategy for bone regeneration: A micro/nano strontium-releasing composite scaffold loaded with lipopolysaccharide

Effective tissue repair relies on the orchestration of different macrophage phenotypes, both the M2 phenotype (promotes tissue repair) and M1 phenotype (pro-inflammatory) deserve attention. In this study, we propose a sequential immune activation strategy to mediate bone regeneration, by loading lipopolysaccharide (LPS) onto the surface of a strontium (Sr) ions -contained composite scaffold, which was fabricated by combining Sr-doped micro/nano-hydroxyapatite (HA) and dual degradable matrices of polycaprolactone (PCL) and poly (lactic-co-glycolic acid) (PLGA). Our strategy involves the sequential release of LPS to promote macrophage homing and induce the expression of the pro-inflammatory M1 phenotype, followed by the release of Sr ions to suppress inflammation. In vitro and in vivo experiments demonstrated that, the appropriate pro-inflammatory effects at the initial stage of implantation, along with the anti-inflammatory effects at the later stage, as well as the structural stability of the scaffolds conferred by the composition, can synergistically promote the regeneration and repair of bone defects.

QS56. Thymosin Alpha 1 Enhances Macrophage Homing and Neutrophil Extracellular Trap Formation in Chronic Diabetic Wound

QS56. Thymosin Alpha 1 Enhances Macrophage Homing and Neutrophil Extracellular Trap Formation in Chronic Diabetic Wound

Nox5 in Vascular Smooth Muscle Cells Mediates Ang II‐Induced Renal Fibrosis and Inflammation

Introduction Mice expressing human Nox5 (hNox5) in vascular smooth muscle cells (VSMC) exhibit increased contraction, ROS generation, cardiac fibrosis and increased pulse pressure. In kidneys, hNox5 in podocytes is associated with albuminuria, hypertension and inflammation. Here we questioned whether VSMC Nox5 influences kidney function and damage in Ang II-induced hypertension. Methods Wildtype (WT) and mice expressing hNOX5 in SMC (Nox5+SM22+) were studied. Mice were infused with Ang II (600 ng/Kg/day) for 28 days. Blood pressure was assessed by tail-cuff, kidney damage by histology, and mRNA and protein expression by qPCR and western-blotting, respectively. Inflammatory infiltrate was assessed by FACS. Results Ang II increased blood pressure (mmHg) in WT (162.7±9) and Nox5+SM22+ (162.3±10). Albuminuria was increased in Nox5+SM22+ versus WT mice (10.2±6 vs 1.27±0.3 mg/mmol, p<0.05) and by Ang II in both groups (Nox5: 44.8±18; WT: 58.7±12 mg/mmol). Kidneys from Ang II-infused Nox5+SM22+ mice exhibited significant perivascular fibrosis and inflammatory cell infiltration compared to WT. Kidney expression of vimentin (AU: 1.01±0.05 vs WT 0.85±0.03) and αSMA (AU: 0.44±0.03 vs WT 0.33±0.01), markers of myofibroblast differentiation, was increased in Nox5 mice (p<0.05), an effect that was augmented by Ang II. In kidneys from Nox5+SM22+ mice, levels of macrophage F4/80+ cells were increased (%: 24±2 vs WT 18±1, p<0.05). Ang II increased macrophage homing in kidneys from WT mice (34±3%, p<0.05 vs non-treated), but did not further increase it in Nox5 mice (35±5%). Levels of cytotoxic CD8+ T cells and expression of IL-1b and IRAK were increased by Ang II in Nox5 mice. Conclusion Our study suggests that Nox5 in VSMCs induces renal fibrosis and pro-inflammatory signalling by Ang II, likely through increased ROS generation.

ROLE OF VASCULAR NOX5 IN ANG II-MEDIATED PRO-FIBROTIC AND PRO-INFLAMMATORY SIGNALLING IN THE KIDNEY

Objective: Mice expressing human Nox5 (hNox5) in vascular smooth muscle cells (VSMC) exhibit increased contraction, ROS generation, cardiac fibrosis and increased pulse pressure. In kidneys, hNox5 in podocytes is associated with albuminuria, hypertension and inflammation. Here we questioned whether VSMC Nox5 influences kidney function and damage in Ang II-induced hypertension. Design and method: Wildtype (WT) and mice expressing hNOX5 in SMC (Nox5+SM22+) were studied. Mice were infused with Ang II (600 ng/Kg/day) for 28 days. Blood pressure was assessed by tail-cuff, kidney damage by histology, and mRNA and protein expression by qPCR and western-blotting, respectively. Inflammatory infiltrate was assessed by FACS. Results: Ang II increased blood pressure (mmHg) in WT (162.7 ± 9) and Nox5+SM22+ (162.3 ± 10). Albuminuria was increased in Nox5+SM22+ versus WT mice (10.2 ± 6 vs 1.27 ± 0.3 mg/mmol, p &lt; 0.05) and by Ang II in both groups (Nox5: 44.8 ± 18; WT: 58.7 ± 12 mg/mmol). Kidneys from Ang II-infused Nox5+SM22+ mice exhibited significant perivascular fibrosis and inflammatory cell infiltration compared to WT. Kidney expression of vimentin (AU: 1.01 ± 0.05 vs WT 0.85 ± 0.03) and aSMA (AU: 0.44 ± 0.03 vs WT 0.33 ± 0.01), markers of myofibroblast differentiation, was increased in Nox5 mice (p &lt; 0.05), an effect that was augmented by Ang II. In kidneys from Nox5+SM22+ mice, levels of macrophage F4/80+ cells were increased (%: 24 ± 2 vs WT 18 ± 1, p &lt; 0.05). Ang II increased macrophage homing in kidneys from WT mice (34 ± 3%, p &lt; 0.05 vs non-treated), but did not further increase it in Nox5 mice (35 ± 5%). Levels of cytotoxic CD8+ T cells and expression of IL-1b and IRAK were increased by Ang II in Nox5 mice. Conclusions: Our study suggests that Nox5 in VSMCs induces renal fibrosis and pro-inflammatory signalling by Ang II, likely through increased ROS generation.

Macrophage membrane functionalized biomimetic nanoparticles for targeted anti-atherosclerosis applications.

Atherosclerosis (AS), the underlying cause of most cardiovascular events, is one of the most common causes of human morbidity and mortality worldwide due to the lack of an efficient strategy for targeted therapy. In this work, we aimed to develop an ideal biomimetic nanoparticle for targeted AS therapy.Methods: Based on macrophage “homing” into atherosclerotic lesions and cell membrane coating nanotechnology, biomimetic nanoparticles (MM/RAPNPs) were fabricated with a macrophage membrane (MM) coating on the surface of rapamycin-loaded poly (lactic-co-glycolic acid) copolymer (PLGA) nanoparticles (RAPNPs). Subsequently, the physical properties of the MM/RAPNPs were characterized. The biocompatibility and biological functions of MM/RAPNPs were determined in vitro. Finally, in AS mouse models, the targeting characteristics, therapeutic efficacy and safety of the MM/RAPNPs were examined.Results: The advanced MM/RAPNPs demonstrated good biocompatibility. Due to the MM coating, the nanoparticles effectively inhibited the phagocytosis by macrophages and targeted activated endothelial cells in vitro. In addition, MM-coated nanoparticles effectively targeted and accumulated in atherosclerotic lesions in vivo. After a 4-week treatment program, MM/RAPNPs were shown to significantly delay the progression of AS. Furthermore, MM/RAPNPs displayed favorable safety performance after long-term administration.Conclusion: These results demonstrate that MM/RAPNPs could efficiently and safely inhibit the progression of AS. These biomimetic nanoparticles may be potential drug delivery systems for safe and effective anti-AS applications.

Inhibition of CD44 attenuates pressure overload-induced cardiac and lung inflammation, fibrosis, and heart failure progression

Abstract Background Inflammation contributes to heart failure (HF) development and progression. CD44 is a member of the hyaluronate receptor family of cell adhesion molecules, which regulates tissue inflammation and fibrosis through modulating macrophage and lymphocyte migration and homing in several diseases. Here we evaluated the role and cellular mechanism of CD44 in regulating transverse aortic constriction (TAC)-induced HF development and progression in mice. Methods and results C57/B6 background CD44 KO and wild type mice (6–8 weeks) were subjected to TAC to evaluate the effect of CD44 on the development of TAC-induced LV hypertrophy and cardiac dysfunction. Due to the rapid response to TAC, Balb/c mice (6–8 weeks) were used to determine the effect of CD44 on the progression of TAC-induced congestive heart failure. We found that CD44 expression is dramatically increased in left ventricular (LV) tissues obtained from HF patients and mice. While CD44 gene knockout (KO) has no detectable effect on cardiac structure and function under control conditions, CD44 KO mice were protected from TAC-induced LV inflammation, fibrosis, hypertrophy, dysfunction, and lung remodeling as compared with wild type mice. In addition, we found that inhibition of CD44 signaling with blocking antibodies (Abs) significantly attenuated the transition from LV failure to lung remodeling, and right ventricular hypertrophy in mice with existing HF. Conclusions These data identify an important role of CD44 in attenuating cardiac and lung inflammation, fibrosis, HF development, and HF progression, suggesting that inhibition of CD44 signaling may be useful in preventing and treating HF. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Chinese National Natural Science Foundation Grants and American Heart Association

BRG1 Stimulates Endothelial Derived Alarmin MRP8 to Promote Macrophage Infiltration in an Animal Model of Cardiac Hypertrophy.

Endothelial cell derived angiocrine factors contribute to the disruption of homeostasis and the pathogenesis of cardiovascular diseases in response to stress stimuli. In the present study we investigated the role of BRG1, a key component of the chromatin remodeling complex, in the regulation of angiocrine signaling. We report that angiotensin II (Ang II) induced pathological cardiac hypertrophy was attenuated in mice with endothelial-specific ablation of BRG1 (ecKO) compared to the control mice (WT). Mitigation of cardiac hypertrophy as a result of BRG1 deficiency was accompanied by decreased macrophage homing to the hearts. This could be explained by the observation that the ecKO mice exhibited down-regulation of myeloid-related protein 8 (MRP8), a well-established chemokine for macrophages, in vascular endothelial cells compared to the WT mice. Further analysis revealed that BRG1 mediated the activation of MRP8 expression by Ang II treatment in endothelial cells to promote macrophage migration. BRG1 was recruited to the MRP8 promoter by interacting with hypoxia-inducible factor 1 (HIF-1α). Reciprocally, BRG1 facilitated the binding of HIF-1α to the MRP8 promoter by sequentially recruiting histone acetyltransferase p300 and histone demethylase KDM3A. Depletion of either p300 or KDM3A repressed the induction of MRP8 expression by Ang II and ameliorated macrophage migration. In conclusion, our data delineate a novel epigenetic pathway whereby Ang II stimulates MRP8 production and macrophage homing to promote cardiac hypertrophy.

Macrophage profile and homing into breast milk in response to ongoing respiratory infections in the nursing infant

Studies have shown that immune components of human milk can be changed during an infection in the nursing infant. Macrophages are abundant in human milk and they are classified into inflammatory (CD16−) and noninflammatory (CD16+) subsets. This study investigated CD16+ and CD16− macrophage homing into breast milk in response to ongoing infections in nursing infants. Peripheral blood and mature milk were collected from 33 healthy mothers of nursing infants with respiratory infections (Group I) and from 26 healthy mothers of healthy nursing infants (Group H). Blood and milk total, CD16− and CD16+ monocyte (Mo)/macrophage (Mφ) subsets, respectively, and CCR2 and CX3CR1 expression and cytokine levels were analyzed by flow cytometry. CCL2 and CX3CL1 were quantified by ELISA and cytokines by flow cytometry in serum and milk. There was an increase of total and CD16+ Mφ, and, also a decrease of CD16- Mφ frequencies in maternal milk from Group I compared to Group H, but absolute numbers analyses showed higher numbers of all subpopulations of milk Mφ in Group I compared to Group H. Higher numbers of CX3CR1+CD16+ and double-staining of CCR2 and CX3CR1 in both CD16+ and CD16− cells were observed in milk during infant infection, which weren’t observed in the blood. CCR2 expression was hardly found in milk CD16− Mφ in both groups. CCL2 and CX3CL1 were both higher in milk than in blood from both groups, but Group I showed higher levels of these chemokines in milk than Group H. Breast milk showed higher IL-6 and IL-8 concentrations than serum, and infant infection caused an increase in these cytokines only in milk. Our findings suggest that milk Mφ profiles are different from blood Mo, and the ongoing infection in the nursing infant could change milk Mφ to a more anti-inflammatory profile compared to that in the healthy group, possibly as an additional strategy of infant protection.

Vessel wall morphology is equivalent for different artery types and localizations of advanced human aneurysms.

Aneurysm formation occurs most frequently as abdominal aortic aneurysm (AAA), but is also seen in other localizations like thoracic or peripheral aneurysm. While initial mechanisms for aneurysm induction remain elusive, observations from AAA samples show transmural inflammation with proteolytic imbalance and repair mechanisms triggered by the innate immune system. However, limited knowledge exists about aneurysm pathology, especially for others than AAA. We compared 42 AAA, 15 popliteal, 3 ascending aortic, five iliac, two femoral, two brachial, one visceral and two secondary aneurysms to non-aneurysmatic controls by histologic analysis, immunohistochemistry and cytokine expression. Muscular and elastic type arteries show a uniform way of aneurysm formation. All samples show similar morphology. The changes compared to controls are distinct and include matrix remodeling with smooth muscle cell phenotype switch and angiogenesis, adventitial lymphoid cell accumulation and M1 macrophage homing together with neutrophil inflammation. Inflammatory cytokines are up-regulated accordingly. Comparative analysis of different disease entities can identify characteristic pathomechanisms. The phenotype of human advanced aneurysm disease is observed for elastic and muscular type arteries, does not differ between disease localizations and might, thus, be a unique response of the vasculature to the still unknown trigger of aneurysm formation.

Factors that promote macrophage homing to adipose tissue in metabolic syndrome

Metabolic syndrome (MetS), a common cardio-metabolic cluster, predisposes to both increased cardiovascular disease and diabetes. Both adipose tissue and monocyte/macrophages contribute to the increased inflammation in MetS. However there are sparse data on factors that determine macrophage recruitment into adipose tissue (AT). In this preliminary report in patients with MetS, without the confounding of diabetes and cardiovascular diseases, we show that plasma resistin, soluble CD14 and monocyte p38 MAP kinase activity correlate significantly with AT macrophage density and hence could be important biomediators of macrophage homing to AT in MetS. However larger studies are required to confirm these novel findings and elucidate other important factors.

Understanding the role of the immune system in adolescent idiopathic scoliosis: Immunometabolic CONnections to Scoliosis (ICONS) study protocol

IntroductionAdolescent idiopathic scoliosis (AIS) affects up to 3% of children around the world. There is limited knowledge of AIS aetiopathogenesis, and this evidence is needed to develop new management strategies....

Persistent infiltration and pro-inflammatory differentiation of monocytes cause unresolved inflammation in brain arteriovenous malformation.

An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68(+) cells at 2weeks (P=0.02), similar numbers at 4weeks (P=0.97), and more at 8weeks (P=0.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8weeks (P=0.064) after angiogenic stimulation and more RFP(+) bone marrow-derived macrophages than WT mice (P=0.01). More CD34(+) cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (P<0.001). These data indicate that persistent infiltration and pro-inflammatory differentiation of monocytes might contribute to macrophage accumulation in bAVM. Blocking macrophage homing to bAVM lesions should be tested as a strategy to reduce the severity of bAVM.

Icmt inhibition exerts anti-angiogenic and anti-hyperpermeability activities impeding malignant pleural effusion.

Small GTPases are pivotal regulators of several aspects of tumor progression. Their implication in angiogenesis, vascular permeability and tumor-associated inflammatory responses is relevant to the pathobiology of Malignant Pleural Effusion (MPE). Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt) abrogates small GTPase activation. We therefore hypothesized that cysmethynil, an Icmt inhibitor would limit pleural fluid accumulation in two models, a lung-adenocarcinoma and a mesothelioma-induced MPE. Cysmethynil significantly reduced MPE volume in both models and tumor burden in the adenocarcinoma model. It inhibited pleural vascular permeability and tumor angiogenesis in vivo and reduced endothelial cell proliferation, migration and tube formation in vitro. Cysmethynil also promoted M1 anti-tumor macrophage homing in the pleural space in vivo, and inhibited tumor-induced polarization of macrophages towards a M2 phenotype in vitro. In addition, the inhibitor promoted adenocarcinoma cell apoptosis in vivo. Inhibition of small GTPase might thus represent a valuable strategy for pharmacotherapy of MPE.

CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury.

Traumatic brain injury (TBI) provokes inflammatory responses, including a dramatic rise in brain macrophages in the area of injury. The pathway(s) responsible for macrophage infiltration of the traumatically injured brain and the effects of macrophages on functional outcomes are not well understood. C-C-chemokine receptor 2 (CCR2) is known for directing monocytes to inflamed tissues. To assess the role of macrophages and CCR2 in TBI, we determined outcomes in CCR2-deficient (Ccr2(-/-)) mice in a controlled cortical impact model. We quantified brain myeloid cell numbers post-TBI by flow cytometry and found that Ccr2(-/-) mice had greatly reduced macrophage numbers (∼80-90% reduction) early post-TBI, compared with wild-type mice. Motor, locomotor, and cognitive outcomes were assessed. Lack of Ccr2 improved locomotor activity with less hyperactivity in open field testing, but did not affect anxiety levels or motor coordination on the rotarod three weeks after TBI. Importantly, Ccr2(-/-) mice demonstrated greater spatial learning and memory, compared with wild-type mice eight weeks after TBI. Although there was no difference in the volume of tissue loss, Ccr2(-/-) mice had significantly increased neuronal density in the CA1-CA3 regions of the hippocampus after TBI, compared with wild-type mice. These data demonstrate that Ccr2 directs the majority of macrophage homing to the brain early after TBI and indicates that Ccr2 may facilitate harmful responses. Lack of Ccr2 improves functional recovery and neuronal survival. These results suggest that therapeutic blockade of CCR2-dependent responses may improve outcomes following TBI.

Macrophages are recruited to hypoxic tumor areas and acquire a pro-angiogenic M2-polarized phenotype via hypoxic cancer cell derived cytokines Oncostatin M and Eotaxin.

TAMs, a unique and distinct M2-skewed myeloid population of tumor stroma, exhibiting pro-tumor functions is fast emerging as a potential target for anti-cancer immunotherapy. Macrophage-recruitment and M2-polarization represent key TAMs-related phenomenon that are amenable to therapeutic intervention. However successful translation of these approaches into effective therapeutic regimen requires better characterization of tumor-microenvironment derived signals that regulate macrophage recruitment and their polarization. Owing to hypoxic milieu being a persistent feature of tumor-microenvironment and a major contributor to malignancy and treatment resistance, the current study was planned with an aim to decipher tumor cell responses to hypoxia vis-a-vis macrophage homing and phenotype switching. Here, we show that hypoxia-primed cancer cells chemoattract and polarize macrophages to pro-angiogenic M2-polarized subtype via Eotaxin and Oncostatin M. Concordantly, hypoxic regions of human breast-cancer specimen exhibited elevated Eotaxin and Oncostatin M levels with concurrently elevated M2-macrophage content. Blockade of Eotaxin/Oncostatin M not only prevented hypoxic breast-cancer cells from recruiting and polarizing macrophages towards an M2-polarized phenotype and retarded tumor progression in 4T1/BALB/c-syngenic-mice-model of breast-cancer but also enhanced the efficacy of anti-angiogenic Bevacizumab. The findings established these two cytokines as novel targets for devising effective anticancer therapy particularly for tumors that are refractory or develop resistance to anti-angiogenic therapeutics.

Endoglin deficiency impairs stroke recovery.

Endoglin deficiency causes hereditary hemorrhagic telangiectasia-1 and impairs myocardial repair. Pulmonary arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia-1 are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that endoglin deficiency impairs stroke recovery. Eng heterozygous (Eng+/-) and wild-type mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Pial collateral vessels were quantified before pMCAO. Infarct/atrophic volume, vascular density, and macrophages were quantified in various days after pMCAO, and behavioral function was assessed using corner and adhesive removal tests on days 3, 15, 30, and 60 after pMCAO. The association between ENG 207G>A polymorphism and brain arteriovenous malformation rupture and surgery outcome was analyzed using logistic regression analysis in 256 ruptured and 157 unruptured patients. After pMCAO, Eng+/- mice showed larger infarct/atrophic volumes at all time points (P<0.05) and showed worse behavior performance (P<0.05) at 15, 30, and 60 days when compared with wild-type mice. Eng+/- mice had fewer macrophages on day 3 (P=0.009) and more macrophages on day 60 (P=0.02) in the peri-infarct region. Although Eng+/- and wild-type mice had similar numbers of pial collateral vessels before pMCAO, Eng+/- mice had lower vascular density in the peri-infarct region (P=0.05) on day 60 after pMCAO. In humans, ENG 207A allele has been associated with worse outcomes after arteriovenous malformation rupture or surgery of patients with unruptured arteriovenous malformation. Endoglin deficiency impairs brain injury recovery. Reduced angiogenesis, impaired macrophage homing, and delayed inflammation resolution could be the underlying mechanism.

Towards delineating the role of dendritic cells and macrophages in SIV disease control and progression (INC9P.447)

Abstract In HIV infection, sustained inflammation and immune activation are signature pathologies of individuals that progress to AIDS. Dendritic cells (DC) and macrophages mediate host inflammatory and antiviral responses and some evidence suggests these cells could be involved in influencing HIV disease outcome, but to what extent is unclear. Here, we longitudinally followed the kinetics of DC and macrophages in peripheral and mucosal tissues from Mamu-B*008+ rhesus macaques intrarectally infected with pathogenic SIVmac251. Mamu-B*008 is a rhesus macaque MHC class I allele highly correlated with control of SIV replication, as greater than 50% of SIV-infected Mamu-B*008+ macaques control chronic phase viral load and do not advance to disease; hence this serves as a model for controlled and progressive HIV disease in humans. We used flow cytometry to access the impact of SIV infection on DC and macrophage proportions, proliferation, and homing to lymph nodes and mucosa. Our preliminary findings reveal a divergent effect of SIV infection on each of the aforementioned DC and macrophage parameters as a function of viral load and tissue compartment. Ongoing experiments will examine the impact of SIV infection on the antigen-presenting cell properties and inflammatory nature of DC and macrophages in the context of SIV disease-progressing and -controlling hosts. These studies should provide clarity on whether DC and macrophages serve a beneficial or detrimental capacity in SIV pathogenesis.

In Vivo Fluorescence-mediated Tomography Imaging Demonstrates Atorvastatin-mediated Reduction of Lesion Macrophages in ApoE-/- Mice

Macrophage recruitment into atherosclerotic plaques drives lesion progression, destabilization, and rupture. Chronic statin treatment reduces macrophage plaque content. Information on dynamics of macrophage recruitment would help assessing plaque vulnerability and guiding therapy. Techniques to image macrophage homing to vulnerable plaques in vivo are scarcely available. The authors tested if noninvasive fluorescence-mediated tomography (FMT) can assess plaque-stabilizing effects of short-term high-dosage atorvastatin. Macrophages from green-fluorescent-protein-transgenic mice were labeled with a near-infrared fluorescent dye and were injected IV in apolipoprotein E-deficient mice (n=9) on Western diet 7 days after guidewire-injury of the carotid artery. FMT-scans, 2 and 7 days thereafter, quantified macrophage recruitment into carotid artery plaques. Atorvastatin was tested for macrophage adhesion, proliferation, and viability (n=5 to 6) in vitro. Fourteen mice received atorvastatin or vehicle for 4 days after 16 weeks on Western diet. FMT assessed macrophage recruitment into aortic and innominate artery lesions. Means (±SD)% are reported. Double-labeled macrophages were recruited into carotid artery lesions. FMT resolved fluorescence projecting on the injured carotid artery and detected a signal increase to 300% (±191) after guidewire injury. Atorvastatin reduced macrophage adhesion to activated endothelial cells by 36% (±19). In a clinically relevant proof-of-concept intervention, FMT-imaging detected that 4 days atorvastatin treatment reduced macrophage recruitment by 57% (±8) indicating plaque stabilization. Immunohistochemistry confirmed reduced macrophage infiltration. FMT optical imaging proved its high potential for clinical applicability for tracking recruitment of near-infrared fluorescent-labeled macrophages to vulnerable plaques in vivo. FMT-based quantification of macrophage recruitment demonstrated rapid plaque stabilization by 4-day atorvastatin treatment in apolipoprotein E-deficient mice.

Abstract TMP69: Endoglin Deficiency Exacerbates Ischemic Brain Injury

Background and Objective: Endoglin (Eng) deficiency causes hereditary hemorrhagic telangiectasia (HHT) and impairs myocardial repair. Pulmonary arteriovenous malformations in HHT patients are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that Eng deficiency exacerbates ischemic brain injury. Methods: Eng heterozygous ( Eng +/- ) mice and wild type (WT) mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Infarct volume and CD68 + cells were quantified 3 days and vascular density was determined 60 days after pMCAO. Behavior was assessed by corner test and adhesive removal test at 3, 15, 30 and 60 days after pMCAO. Matrix metalloproteinase 9 (Mmp9) and Notch1 expression in bone marrow (BM)-derived macrophages from Eng +/- and WT were analyzed using real-time RT-PCR. Results: Eng +/- mice had a larger Infarct volume than WT mice (22±6% of the affected hemisphere vs. 16±6%, p=0.04). Eng +/- mice had longer adhesive-removal time (p&lt;0.05) and more frequent turning to the lesion side than WT mice at 15, 30 and 60 days (p&lt;0.05) after pMCAO. Both groups had similar numbers of CD68 + cells in the peri-infarct area at 3 days after pMCAO (370±80 vs 338±44 cells/mm 2 , p=0.37), but Eng +/- mice had lower peri-infarct vessel density (417±69 vs 490±52 vessels/mm 2 , p=0.05) at 60 days after pMCAO. Up-regulation of Mmp9 and Notch1 expression in response to VEGF was attenuated in Eng +/- BM-derived macrophages. Conclusions: Endoglin deficiency exacerbated brain injury and behavior dysfunction in mice after pMCAO and was associated with reduced angiogenesis. Although macrophage homing was not affected, reduced expression of two angiogenic-related genes, Mmp9 and Notch1 , by Eng +/- BM-derived macrophages suggests a potential role of these cells in recovery from an ischemic injury.

Bioluminescence and Magnetic Resonance Imaging of Macrophage Homing to Experimental Abdominal Aortic Aneurysms

Macrophage infiltration is a prominent feature of abdominal aortic aneurysm (AAA) progression. We used a combined imaging approach with bioluminescence (BLI) and magnetic resonance imaging (MRI) to study macrophage homing and accumulation in experimental AAA disease. Murine AAAs were created via intra-aortic infusion of porcine pancreatic elastase. Mice were imaged over 14 days after injection of prepared peritoneal macrophages. For BLI, macrophages were from transgenic mice expressing luciferase. For MRI, macrophages were labeled with iron oxide particles. Macrophage accumulation during aneurysm progression was observed by in situ BLI and by in vivo 7T MRI. Mice were sacrificed after imaging for histologic analysis. In situ BLI (n = 32) demonstrated high signal in the AAA by days 7 and 14, which correlated significantly with macrophage number and aortic diameter. In vivo 7T MRI (n = 13) at day 14 demonstrated T₂* signal loss in the AAA and not in sham mice. Immunohistochemistry and Prussian blue staining confirmed the presence of injected macrophages in the AAA. BLI and MRI provide complementary approaches to track macrophage homing and accumulation in experimental AAAs. Similar dual imaging strategies may aid the study of AAA biology and the evaluation of novel therapies.