Impact of Femoral Ossification on Local and Systemic Cardiovascular Patients' Condition.

BACKGROUND: Vascular calcifications are associated with a high cardiovascular morbi-mortality in the coronary territory. In parallel, femoral arteries are more calcified and develop osteoid metaplasia (OM). This study was conducted to assess the predictive value of OM and local inflammation on the occurrence of mid- and long-term adverse cardiovascular events. METHOD: Between 2008 and 2015, 86 atheromatous samples were harvested during femoral endarterectomy on 81 patients and processed for histomorphological analyses of calcifications and inflammation (monocytes and B cells). Histological findings were compared with the long-term follow-up of patients, including major adverse cardiac event (MACE), major adverse limb event (MALE), and mortality. Frequencies were presented as percentage, and continuous data, as mean and standard deviation. A P-value < 0.05 was considered statistically significant. RESULTS: Median follow-up was 42.4 months (26.9-58.8). Twenty-eight percent of patients underwent a MACE; a MALE occurred in 18 (21%) limbs. Survival rate was 87.2% at 36 months. OM was found in 41 samples (51%), without any significant impact on the occurrence of MACE, MALE, or mortality. Preoperative white blood cell formulae revealed a higher rate of neutrophils associated with MACE (P = 0.04) and MALE (P = 0.0008), correlated with higher B cells counts in plaque samples. CONCLUSIONS: OM is part of femoral calcifications in almost 50% of the cases but does not seem to be an independent predictive variable for MACE or MALE. However, a higher rate of B cell infiltration of the plaque and preoperative neutrophil blood count may be predictive of adverse events during follow-up.

BLTR1 in Monocytes Emerges as a Therapeutic Target For Vascular Inflammation With a Subsequent Intimal Hyperplasia in a Murine Wire-Injured Femoral Artery.

Given the importance of high-mobility group box 1 (HMGB1) and 5-lipoxygenase (5-LO) signaling in vascular inflammation, we investigated the role of leukotriene signaling in monocytes on monocyte-to-macrophage differentiation (MMD) induced by HMGB1, and on vascular inflammation and subsequent intimal hyperplasia in a mouse model of wire-injured femoral artery. In cultured primary bone marrow-derived cells (BMDCs) stimulated with HMGB1, the number of cells with macrophage-like morphology was markedly increased in association with an increased expression of CD11b/Mac-1, which were attenuated in cells pre-treated with Zileuton, a 5-LO inhibitor as well as in 5-LO-deficient BMDCs. Of various leukotriene receptor inhibitors examined, which included leukotriene B4 receptors (BLTRs) and cysteinyl leukotriene receptors (cysLTRs), the BLTR1 inhibitor (U75302) exclusively suppressed MMD induction by HMGB1. The importance of BLTR1 in HMGB1-induced MMD was also observed in BMDCs isolated from BLTR1-deficient mice and BMDCs transfected with BLTR1 siRNA. Although leukotriene B4 (LTB4) had minimal direct effects on MMD in control and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was significantly reversed by exogenous LTB4, but not in BLTR1-deficient BMDCs, suggesting that LTB4/BLTR1-mediated priming of monocytes is a prerequisite of HMGB1-induced MMD. In vivo, both macrophage infiltration and intimal hyperplasia in our wire-injured femoral artery were markedly attenuated in BLTR1-deficient mice as compared with wild-type controls, but these effects were reversed in BLTR1-deficient mice transplanted with monocytes from control mice. These results suggest that BLTR1 in monocytes is a pivotal player in MMD with subsequent macrophage infiltration into neointima, leading to vascular remodeling after vascular injury.

Distinct Differences on Neointima Formation in Immunodeficient and Humanized Mice after Carotid or Femoral Arterial Injury.

Percutaneous coronary intervention is widely adopted to treat patients with coronary artery disease. However, restenosis remains an unsolved clinical problem after vascular interventions. The role of the systemic and local immune response in the development of restenosis is not fully understood. Hence, the aim of the current study was to investigate the role of the human immune system on subsequent neointima formation elicited by vascular injury in a humanized mouse model. Immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl(NSG) mice were reconstituted with human (h)PBMCs immediately after both carotid wire and femoral cuff injury were induced in order to identify how differences in the severity of injury influenced endothelial regeneration, neointima formation, and homing of human inflammatory and progenitor cells. In contrast to non-reconstituted mice, hPBMC reconstitution reduced neointima formation after femoral cuff injury whereas hPBMCs promoted neointima formation after carotid wire injury 4 weeks after induction of injury. Neointimal endothelium and smooth muscle cells in the injured arteries were of mouse origin. Our results indicate that the immune system may differentially respond to arterial injury depending on the severity of injury, which may also be influenced by the intrinsic properties of the arteries themselves, resulting in either minimal or aggravated neointima formation.

A depleting antibody toward sca-1 mitigates a surge of CD34(+)/c-kit(+) progenitors and reduces vascular restenosis in a murine vascular injury model.

OBJECTIVE: Vascular restenosis remains a major obstacle to long-term success after vascular intervention. Circulating progenitor cells have been implicated in restenosis, and yet it has remained unclear if these cells, particularly nonendothelial progenitors, have an active role in this pathologic process. We hypothesized that circulating CD34(+)/c-kit(+) progenitors would increase after vascular injury, mirrored by changes in the injury signal, stromal cell-derived factor 1α (sdf1α). We further postulated that an antibody-based depletion would mitigate progenitor surge and, in turn, reduce restenosis in a murine model. METHODS: C57BL6 mice underwent wire injury of the femoral artery and were compared with mice with sham surgery and vessel ligation by flow cytometry as well as by sdf1α enzyme-linked immunosorbent assay of peripheral blood. Next, injured C57BL6 mice treated with a depleting antibody toward the progenitor marker sca-1 or with an isotype control were compared in terms of sdf1α as well as enumeration of progenitors. At 28 days, restenosis was quantified between sca-1- and isotype-treated animals. RESULTS: Wire injury generated an increase in sdf1α as well as a surge of CD34(+)/c-kit(+) progenitors relative to nonsurgical controls (P = .005). Treatment with sca-1 antibody ablated the peripheral surge compared with isotype-treated, injured animals (P = .02), and sca progenitor depletion reduced the 28-day intima to media ratio in a statistically significant fashion compared with either nontreated (P = .04) or isotype-treated (P = .036) animals. CONCLUSIONS: Our study has demonstrated that sca-1 antibody reduces both progenitor surge and vascular restenosis after endoluminal vascular injury in a murine model. This suggests that circulating progenitors play an active role in restenotic disease.

Increased Number of Mast Cells in Atherosclerotic Lesions Correlates with the Presence of Myeloid but not Plasmacytoid Dendritic Cells as well as Pro-inflammatory T Cells.

BACKGROUND: Atherosclerosis is an inflammatory disease of the vessel wall promoted by different immune cells and inflammatory mediators. METHODS: In this study, 26 human plaques and 12 control vessels without atherosclerosis were immunohistochemically stained to analyze the emergence of mast cells dependent on plaque morphology and to correlate mast cell occurrence with the emergence of myeloid as well as plasmacytoid dendritic cells. Also, mast cell emergence was correlated with the number of pro-inflammatory T cells. For this, plaques were classified as stable or unstable according to established histological criteria. RESULTS: As expected, atherosclerotic lesions showed significantly higher numbers of tryptase+, chymase+, and cathepsin G+ mast cells compared to control vessels, particularly in lesions with unstable morphology. As a novel finding, we detected significant correlations between mast cells and myeloid dendritic cells (fascin, CD83, r > 0.3, p < 0.01), but not plasmacytoid dendritic cells (CD123, CD304). Also, we observed significant correlations of mast cells and different subgroups of pro-inflammatory T cells (CD3, CD8, CD161, CD25; r > 0.35, p < 0.05). CONCLUSIONS: Overall, the higher number of mast cells in plaques, particularly with unstable morphology, suggests that mast cells might be involved in the progression of atherosclerosis. The correlation of mast cells with other immune cells that are pivotal in atherogenesis, e.g., myeloid dendritic cells and pro-inflammatory T cells, also suggests an interplay leading to plaque destabilization. Therefore, modulating local mast cell function and invasion into the plaque might be a therapeutic tool for plaque stabilization.

CD4(+) lymphocytes improve venous blood flow in experimental arteriovenous fistulae.

BACKGROUND: The role of immune cells in arteriovenous fistulae (AVF) maturation is poorly understood and has received, until quite recently, little attention. This study examines the function of T lymphocytes in AVF vascular remodeling. METHODS: Experimental fistulae were created in athymic rnu nude rats lacking mature T lymphocytes and euthymic control animals by anastomosing the left superior epigastric vein to the nearby femoral artery. Blood flow rates, wall morphology, and histologic changes were assessed in AVF 21 days after creation. The effect of CD4(+) lymphocytes on AVF maturation in athymic animals was analyzed by adoptive transfer of cells after fistula creation. RESULTS: The absence of T lymphocytes compromised blood flow in experimental fistulae. Histopathologic inspection of AVF from athymic rats revealed that T-cell immunodeficiency negatively affected venous vascular remodeling, as evidenced by a reduced lumen, a thick muscular layer, and a low number of inflammatory cells compared with control animals. Adoptive transfer of CD4(+) lymphocytes from euthymic rats into athymic animals after fistula creation improved blood flow and reduced intima-media thickness. CONCLUSION: These results point at the protective role of CD4(+) lymphocytes in the remodeling of the AVF vascular wall.

MAb therapy against the IFN-α/ß receptor subunit 1 stimulates arteriogenesis in a murine hindlimb ischaemia model without enhancing atherosclerotic burden.

AIMS: IFN-beta (IFNß) signalling is increased in patients with insufficient coronary collateral growth (i.e. arteriogenesis) and IFNß hampers arteriogenesis in mice. A downside of most pro-arteriogenic agents investigated in the past has been their pro-atherosclerotic properties, rendering them unsuitable for therapeutic application. Interestingly, type I IFNs have also been identified as pro-atherosclerotic cytokines and IFNß treatment increases plaque formation and accumulation of macrophages. We therefore hypothesized that mAb therapy to inhibit IFNß signalling would stimulate arteriogenesis and simultaneously attenuate-rather than aggravate-atherosclerosis. METHODS AND RESULTS: In a murine hindlimb ischaemia model, atherosclerotic low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated during a 4-week period with blocking MAbs specific for mouse IFN-α/ß receptor subunit 1 (IFNAR1) or murine IgG isotype as a control. The arteriogenic response was quantified using laser Doppler perfusion imaging (LDPI) as well as immunohistochemistry. Effects on atherosclerosis were determined by quantification of plaque area and analysis of plaque composition. Downstream targets of IFNß were assessed by real-time PCR (RT-PCR) in the aortic arch. Hindlimb perfusion restoration after femoral artery ligation was improved in mice treated with anti-IFNAR1 compared with controls as assessed by LDPI. This was accompanied by a decrease in CXCL10 expression in the IFNAR1 MAb-treated group. Anti-IFNAR1 treatment reduced plaque apoptosis without affecting total plaque area or other general plaque composition parameters. Results were confirmed in a short-term model and in apolipoprotein E knockout (APOE)(-/-) mice. CONCLUSION: Monoclonal anti-IFNAR1 therapy during a 4-week treatment period stimulates collateral artery growth in mice and did not enhance atherosclerotic burden. This is the first reported successful strategy using MAbs to stimulate arteriogenesis.

Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response.

Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.

Porphyromonas gingivalis promotes neointimal formation after arterial injury through toll-like receptor 2 signaling.

We previously demonstrated that Porphyromonas gingivalis infection induces neointimal hyperplasia with an increase in monocyte chemoattractant protein (MCP)-1 after arterial injury in wild-type mice. Toll-like receptor (TLR) 2 is a key receptor for the virulence factors of P. gingivalis. The aim of this study was to assess whether TLR2 plays a role in periodontopathic bacteria-induced neointimal formation after an arterial injury. Wild-type and TLR2-deficient mice were used in this study. The femoral arteries were injured, and P. gingivalis or vehicle was injected subcutaneously once per week. Fourteen days after arterial injury, the murine femoral arteries were obtained for histopathologic and immunohistochemical analyses. The immunoglobulin-G levels of the P. gingivalis-infected groups were significantly increased in comparison with the level in the corresponding noninfected groups in both wild-type and TLR2-deficient mice. TLR2 deficiency negated the P. gingivalis-induced neointimal formation in comparison with the wild-type mice, and reduced the number of positive monocyte chemoattractant protein-1 cells in the neointimal area. These findings demonstrate that P. gingivalis infection can promote neointimal formation after an arterial injury through TLR2 signaling.

Identification of inflamed atherosclerotic lesions in vivo using PET-CT.

Inflammation plays a major pathogenetic role in the development of atherosclerotic plaques and related thromboembolic events. The identification of vulnerable plaques is of the utmost importance, as this may allow the implementation of more effective preventive and therapeutic interventions. Fluorodeoxyglucose positron emission tomography (FDG-PET) has been shown to be useful for tracing inflammation within plaques. However, its relationship to immunohistochemical findings in different territories of the peripheral circulation was not completely elucidated. We aimed to determine whether plaque inflammation could be measured by PET in combination with computer tomography (CT) using FDG and what is the relationship between FDG uptake and immunohistochemical findings in the removed atherosclerotic lesions of the femoral and carotid arteries. The study included 31 patients, 21 patients with high-grade stenosis of the internal carotid artery (ICA) and 10 patients with occlusion of the common femoral artery (CFA), all of whom underwent endarterectomy. Before endarterectomy in all patients, FDG-PET/CT imaging was performed. FDG uptake was measured as the maximum blood--normalized standardized uptake value, known as the target to background ratio (TBR max). TBR max amounted to 1.72 ± 0.8, and in patients with ICA, stenosis was not significantly different from patients with CFA occlusion. Immunohistochemical and morphometric analyses of the plaques obtained at endarterectomy showed that the density of T lymphocytes and macrophages (number of cells per square millimeter) was significantly higher in subjects with stenosis of the ICA than in subjects with occlusion of the femoral arteries: lymphocytes, 1.26 ± 0.21 vs. 0.77 ± 0.29; p = 0.02 and macrophages, 1.01 ± 0.18 vs. 0.69 ± 0.23; p = 0.003. In the whole group of patients, the density of inflammatory cells significantly correlated with FDG uptake represented by PET-TBR max: T lymphocytes, r = 0.60; p < 0.01 and macrophages, r = 0.65; p < 0.01. The results of our study show that FDG uptake is related to the accumulation of inflammatory cells in atherosclerotic lesions. This finding suggests that FDG uptake reflects the severity of atherosclerotic vessel wall inflammation, and in stenotic lesions, it could be an indicator of their vulnerability. However, data from large outcome studies is needed to estimate the usefulness of this technique in identifying the most dangerous atherosclerotic lesions and vulnerable patients.

Prostaglandin E2 potentiates mesenchymal stem cell-induced IL-10+IFN-γ+CD4+ regulatory T cells to control transplant arteriosclerosis.

Mesenchymal stem cells (MSCs) are known for their immunomodulatory functions. We previously demonstrated that bone marrow-derived MSCs effectively control transplant arteriosclerosis (TA) by enhancing IL-10(+) and IFN-γ(+) cells. The objective of this study is to elucidate the mechanism by which MSCs induce IL-10(+)IFN-γ(+)CD4(+) regulatory T type 1 (T(R)1)-like cells. In an MLR system using porcine PBMCs, MSC-induced IL-10(+)IFN-γ(+)CD4(+) cells, which confer resistance to allogeneic proliferation in an IL-10-dependent manner, resemble T(R)1-like cells. Both cyclooxygenase-derived PGE(2) and IDO help to induce T(R)1-like cells by MSCs. MSCs constitutively secrete PGE(2), which is augmented in allogeneic reactions. However, T(R)1-like cells were deficient in PGE(2) and 4-fold less potent than were MSCs in suppressing MLR. PGE(2) mimetic supplements can enhance the immunosuppressive potency of T(R)1-like cells. In a porcine model of allogeneic femoral arterial transplantation, MSC-induced T(R)1-like cells combined with PGE(2), but not either alone, significantly reduced TA at the end of 6 wk (percentage of luminal area stenosis: T(R)1-like cells + PGE(2): 11 ± 10%; PGE(2) alone: 93 ± 8.7%; T(R)1-like cells alone: 88 ± 2.4% versus untreated 94 ± 0.9%, p < 0.001). These findings indicate that PGE(2) helps MSC-induced IL-10(+)IFN-γ(+)CD4(+) T(R)1-like cells inhibit TA. PGE(2) combined with MSC-induced T(R)1-like cells represents a new approach for achieving immune tolerance.

T-cell co-stimulation by CD28-CD80/86 and its negative regulator CTLA-4 strongly influence accelerated atherosclerosis development.

OBJECTIVE: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. METHODS AND RESULTS: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4(-/-) and CD80(-/-)CD86(-/-) mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p=0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p=0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1+, PD1+, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. CONCLUSIONS: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.

Importance of endothelial NF-κB signalling in vascular remodelling and aortic aneurysm formation.

AIMS: Vascular remodelling and aortic aneurysm formation are induced mainly by inflammatory responses in the adventitia and media. However, relatively little is known about the mechanistic significance of endothelium in the pathogenesis of these vascular disorders. The transcription factor nuclear factor-kappa B (NF-κB) regulates the expressions of numerous genes, including those related to pro-inflammatory responses. Therefore, to investigate the roles of endothelial pro-inflammatory responses, we examined the impact of blocking endothelial NF-κB signalling on intimal hyperplasia and aneurysm formation. METHODS AND RESULTS: To block endothelial NF-κB signalling, we used transgenic mice expressing dominant-negative IκBα selectively in endothelial cells (E-DNIκB mice). E-DNIκB mice were protected from the development of cuff injury-induced neointimal formation, in association with suppressed arterial expressions of cellular adhesion molecules, a macrophage marker, and inflammatory factors. In addition, the blockade of endothelial NF-κB signalling prevented abdominal aortic aneurysm formation in an experimental model, hypercholesterolaemic apolipoprotein E-deficient mice with angiotensin II infusion. In this aneurysm model as well, aortic expressions of an adhesion molecule, a macrophage marker, and inflammatory factors were suppressed with the inhibited expression and activity of matrix metalloproteinases in the aorta. CONCLUSION: Endothelial NF-κB activation up-regulates adhesion molecule expression, which may trigger macrophage infiltration and inflammation in the adventitia and media. Thus, the endothelium plays important roles in vascular remodelling and aneurysm formation through its intracellular NF-κB signalling.

High-mobility-group box protein 1A box reduces development of sodium laurate-induced thromboangiitis obliterans in rats.

OBJECTIVE: High-mobility-group box protein 1 (HMGB1), as a late mediator of inflammation, plays a key role in inflammatory responses by inducing and extending the production of proinflammatory cytokines. The effect of HGMB1 in the inflammatory disease thromboangiitis obliterans (TAO) is unknown. We aimed to investigate the role of HMGB1 in sodium laurate-induced TAO in rats. METHODS: Male Wistar rats were randomly divided into five groups (n=8 each) for treatment: normal, sham-operated, TAO model, and low-dose (15 mg/kg) or high-dose (30 mg/kg) recombinant A box (rA box) infection (administered intraperitoneally once daily for 15 days). The TAO model was induced by sodium laurate and graded by gross appearance on day 15 after femoral artery injection. Histologic changes were measured by histopathology in rat femoral arteries. Plasma levels of HMGB1, thromboxane B2, 6-keto-prostaglandin F1-α, and blood cell counts and blood coagulation levels were measured. Expression of HMGB1, receptor for advanced glycation end-products (RAGE), interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was assessed by immunohistochemistry and immunofluorescence, Western blot analysis, and quantitative reverse-transcription polymerase chain reaction. RESULTS: The typical signs and symptoms of TAO were observed on day 15 after sodium laurate injection. The expression of HMGB1, RAGE, interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was markedly increased in rat femoral arteries. Plasma levels of HMGB1 and thromboxane B2 were elevated, but the level of 6-keto-prostaglandin F1-α was decreased. Blood was in a hypercoagulable state, and prothrombin, thrombin, and activated partial thromboplastin times were all significantly shortened, whereas fibrinogen level was increased in TAO rats compared with sham-operated rats. These effects were terminated by the HMGB1 antagonist rA box. CONCLUSIONS: HMGB1 is involved in the inflammatory state in a model of TAO induced by sodium laurate in rats, probably via its receptor RAGE. As the antagonist of HMGB1, rA box can attenuate the development of TAO, which may be a potential therapeutic target for the treatment of TAO.

A clinicopathologic study of immunoglobulin G4-related disease of the femoral and popliteal arteries in the spectrum of immunoglobulin G4-related periarteritis.

BACKGROUND: Immunoglobulin (Ig) G4-related disease has recently been recognized to occur in the cardiovascular system in the aorta and main branching arteries, often manifesting as aneurysms and arteritis/periarteritis. Peripheral arteries (the femoral and popliteal arteries) are frequent sites of arteriosclerosis obliterans (ASO) and occasionally show aneurysms or arteritis. This study re-examined peripheral arterial lesions from the standpoint of IgG4-related disease. METHODS: The study comprised 104 patients who underwent surgical treatment of peripheral arterial lesions, including 30 patients with peripheral arterial aneurysms (PAAs) and 74 with ASO. IgG4-related disease was identified on the basis of diffuse infiltration of numerous IgG4-positive plasmacytes as revealed by immunohistochemical examination. Clinicopathologic features were compared between IgG4-related and IgG4-unrelated lesions. RESULTS: IgG4-related disease was found in four of the 30 patients with PAAs (13.3%; two in the deep femoral artery, two in the popliteal artery) but not in any patients with ASO. IgG4-related PAA displayed clinicopathologic features resembling those of other IgG4-related diseases and a characteristic saccular appearance (P = .002). CONCLUSIONS: IgG4-related disease was detected in PAA patients but not in ASO patients. IgG4-related disease thus represents one potential etiology of aneurysm in the peripheral arteries.

Skin own bacteria may aggravate inflammatory and occlusive changes in atherosclerotic arteries of lower limbs.

AIM: Seroepidemiological studies have given rise to the hypothesis that microorganisms like Chlamydia pneumoniae (CP), Helicobacter pylori (HP), cytomegalovirus (CMV), HCV types 1 and 2, and bacteria involved in dental or other unspecified infection sites may initiate or maintain the atherosclerotic process in lower limb arteries. However, not much attention has been attached to the patient's own limb skin and deep tissues bacterial flora, activated in ischemic tissues. This flora may enhance the inflammatory and thrombotic process in the atherosclerotic arteries. Lower limb tissues are exposed to microorganisms from the environment (foot) and microbes on floating epidermal cells from the perineal and anal regions. The aim of this paper was to identify microbial cells and their DNA in perivascular tissues and arterial walls of lower limbs. METHODS: Bacterial cultures and PCR method for detection of 16sRNA and immunohistopathological staining for identification of immune cells infiltrating vascular bundles. RESULTS: 1) specimens of atherosclerotic calf and femoral arteries contained bacterial isolates and/or their DNA, whereas, in control normal cadaveric organ donors' limb arteries or patients' carotid arteries and aorta bacteria they were detected only sporadically; 2) lower limb lymphatics contained bacterial cells in 76% of specimens, whereas controls only in 10%; 3) isolates from limb arteries and lymphatics belonged in majority to the coagulase-negative staphylococci and S.aureus, however, other highly pathogenic strains were also detected; 4) immunohistopathological evaluation arterial walls showed dense focal infiltrates of granulocytes and macrophages. CONCLUSION: Own bacterial isolates can be responsible for dense neutrophil and macrophage inflitrates of atherosclerotic walls and periarterial tissue in lower limbs and aggravate the ischemic changes.

Macrophage subtypes in symptomatic carotid artery and femoral artery plaques.

OBJECTIVE: To compare differences in macrophage heterogeneity and morphological composition between atherosclerotic plaques obtained from recently symptomatic patients with carotid artery disease and femoral plaques from patients with severe limb ischemia. DESIGN: Experimental study. METHODS: Plaques were obtained from 32 patients undergoing carotid endarterectomy and 25 patients undergoing common femoral endarterectomy or lower limb bypass. Macrophages and T cell numbers were detected in plaque sections by immunohistochemistry and anti CD68 and CD3 antibodies. Dual staining for CD68 and M1- and M2-macrophage markers and morphometric analysis of hematoxylin and eosin stained plaque sections was performed. RESULTS: Carotid plaques had significantly increased percentage areas of confluent lipid and leukocytic infiltrates. In contrast, areas of fibroconnective tissue were significantly greater in femoral plaques and percentage areas of confluent calcification and collagen were elevated. Carotid artery plaques had greater numbers per plaque area of macrophages and T cells consistent with a more inflammatory phenotype. Proportions displaying M1-activation markers were significantly increased in the carotid compared to femoral plaques whereas femoral plaques displayed a greater proportion of M2-macrophages. CONCLUSION: Plaques from patients with recently symptomatic carotid disease have a predominance of M1-macrophages and higher lipid content than femoral plaques, consistent with a more unstable plaque.

Evaluation of the humoral and cellular immune responses after implantation of a PTFE vascular prosthesis.

INTRODUCTION: The experiment was designed in order to determine the immunological processes that occur during the healing in synthetic vascular grafts, especially to establish the differences in the location of the complement system proteins between the proximal and distal anastomosis and the differences in the arrangement of inflammatory cells in those anastomoses. The understanding of those processes will provide a true basis for determining risk factors for complications after arterial repair procedures. MATERIAL/METHODS: The experiment was carried out on 16 dogs that underwent implantation of unilateral aorto-femoral bypass with expanded polytetrafluoroethylene (ePTFE). After 6 months all animals were euthanized to dissect the vascular grafts. Immunohistochemical assays and electron microscopic examinations were performed. RESULTS: Immunohistochemical findings in the structure of neointima between anastomoses of vascular prostheses demonstrated significant differences between humoral and cellular responses. The area of proximal anastomosis revealed the presence of fibroblasts, but no macrophages were detected. The histological structure of the proximal anastomosis indicates that inflammatory processes were ended during the prosthesis healing. The immunological response obtained in the distal anastomosis corresponded to the chronic inflammatory reaction with the presence of macrophages, myofibroblasts and deposits of complement C3. DISCUSSION: The identification of differences in the presence of macrophages and myofibroblasts and the presence of the C3 component between the anastomoses is the original achievement of the present study. In the available literature, no such significant differences have been shown so far in the humoral and cellular immune response caused by the presence of an artificial vessel in the arterial system.

Correlation between arterial FDG uptake and biomarkers in peripheral artery disease.

OBJECTIVES: A prospective, multicenter (18)fluorine-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET)/computed tomography (CT) imaging study was performed to estimate the correlations among arterial FDG uptake and atherosclerotic plaque biomarkers in patients with peripheral artery disease. BACKGROUND: Inflammation within atherosclerotic plaques is associated with instability of the plaque and future cardiovascular events. Previous studies have shown that (18)F-FDG-PET/CT is able to quantify inflammation within carotid artery atherosclerotic plaques, but no studies to date have investigated this correlation in peripheral arteries with immunohistochemical confirmation. METHODS: Thirty patients across 5 study sites underwent (18)F-FDG-PET/CT imaging before SilverHawk atherectomy (FoxHollow Technologies, Redwood City, California) for symptomatic common or superficial femoral arterial disease. Vascular FDG uptake (expressed as target-to-background ratio) was measured in the carotid arteries and aorta and femoral arteries, including the region of atherectomy. Immunohistochemistry was performed on the excised atherosclerotic plaque extracts, and cluster of differentiation 68 (CD68) level as a measure of macrophage content was determined. Correlations between target-to-background ratio of excised lesions, as well as entire arterial regions, and CD68 levels were determined. Imaging was performed during the 2 weeks before surgery in all cases. RESULTS: Twenty-one patients had adequate-quality (18)F-FDG-PET/CT peripheral artery images, and 34 plaque specimens were obtained. No significant correlation between lesion target-to-background ratio and CD68 level was observed. CONCLUSIONS: There were no significant correlations between CD68 level (as a measure of macrophage content) and FDG uptake in the peripheral arteries in this multicenter study. Differences in lesion extraction technique, lesion size, the degree of inflammation, and imaging coregistration techniques may have been responsible for the failure to observe the strong correlations with vascular FDG uptake observed in previous studies of the carotid artery and in several animal models of atherosclerosis.