Successful immunosuppressant-free heterotopic transplantation of tracheal allografts in the pig.

OBJECTIVES: It has been demonstrated that both heterotopic and orthotopic transplants of epithelium-denuded cryopreserved tracheal allografts are feasible in immunosuppressant-free rabbits. Validation of these results in large animals is required before considering clinical applications. We evaluated the viability, immune tolerance and strain properties of such tracheal allografts heterotopically transplanted in a pig model. METHODS: Ten tracheal segments, 5 short (5 rings) and 5 long (10 rings), were obtained from male Landrace pigs. The tracheal segments were surgically denuded of their epithelium, then cryopreserved and stored in a tissue bank for 33 to 232 days. After thawing, tracheal segments stented with a silicone tube were wrapped in the omentum in 2 groups of 5 female recipients. The animals did not receive any immunosuppressive drugs. The animals were euthanized from Day 6 to Day 90 in both groups. RESULTS: An effective revascularization of allografts regardless of length was observed. Lymphocyte infiltrate was shown in the early postoperative period and became non-significant after 30 days. Allografts displayed high levels of neoangiogenesis and viable cartilage rings with islets of calcification. Biomechanical measurements demonstrated strain properties similar to those of a fresh tracheal segment from Day 58. CONCLUSIONS: Our results demonstrate the acceptability and satisfactory stiffness of epithelium-denuded cryopreserved tracheal allografts implanted in the omentum, despite the absence of immunosuppressive drugs. Since the omentum has the capability to reach the tracheal region, this approach should be investigated in the setting of orthotopic transplants in a pig model before considering clinical applications.

Human Alternative Macrophages Populate Calcified Areas of Atherosclerotic Lesions and Display Impaired RANKL-Induced Osteoclastic Bone Resorption Activity.

RATIONALE: Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. OBJECTIVE: To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. METHODS AND RESULTS: Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos-mediated downstream signaling resulting in impaired bone resorption capacity. CONCLUSIONS: These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.

Von Willebrand Factor Multimers during Transcatheter Aortic-Valve Replacement.

BACKGROUND: Postprocedural aortic regurgitation occurs in 10 to 20% of patients undergoing transcatheter aortic-valve replacement (TAVR) for aortic stenosis. We hypothesized that assessment of defects in high-molecular-weight (HMW) multimers of von Willebrand factor or point-of-care assessment of hemostasis could be used to monitor aortic regurgitation during TAVR. METHODS: We enrolled 183 patients undergoing TAVR. Patients with aortic regurgitation after the initial implantation, as identified by means of transesophageal echocardiography, underwent additional balloon dilation to correct aortic regurgitation. HMW multimers and the closure time with adenosine diphosphate (CT-ADP), a point-of-care measure of hemostasis, were assessed at baseline and 5 minutes after each step of the procedure. Mortality was evaluated at 1 year. A second cohort (201 patients) was studied to validate the use of CT-ADP in order to identify patients with aortic regurgitation. RESULTS: After the initial implantation, HMW multimers normalized in patients without aortic regurgitation (137 patients). Among the 46 patients with aortic regurgitation, normalization occurred in 20 patients in whom additional balloon dilation was successful but did not occur in the 26 patients with persistent aortic regurgitation. A similar sequence of changes was observed with CT-ADP. A CT-ADP value of more than 180 seconds had sensitivity, specificity, and negative predictive value of 92.3%, 92.4%, and 98.6%, respectively, for aortic regurgitation, with similar results in the validation cohort. Multivariable analyses showed that the values for HMW multimers and CT-ADP at the end of TAVR were each associated with mortality at 1 year. CONCLUSIONS: The presence of HMW-multimer defects and a high value for a point-of-care hemostatic test, the CT-ADP, were each predictive of the presence of aortic regurgitation after TAVR and were associated with higher mortality 1 year after the procedure. (Funded by Lille 2 University and others; ClinicalTrials.gov number, NCT02628509.).

A novel ELISA-based diagnosis of acquired von Willebrand disease with increased VWF proteolysis.

Von Willebrand disease-type 2A (VWD-2A) and acquired von Willebrand syndrome (AVWS) due to aortic stenosis (AS) or left ventricular assist device (LVAD) are associated with an increased proteolysis of von Willebrand factor (VWF). Analysis of VWF multimeric profile is the most sensitive way to assess such increased VWF-proteolysis. However, several technical aspects hamper a large diffusion among routine diagnosis laboratories. This makes early diagnosis and early appropriate care of increased proteolysis challenging. In this context of unmet medical need, we developed a new ELISA aiming a quick, easy and reliable assessment of VWF-proteolysis. This ELISA was assessed successively in a LVAD-model, healthy subjects (n=39), acquired TTP-patients (n=4), VWD-patients (including VWD-2A(IIA), n=22; VWD-2B, n=26; VWD-2A(IIE), n=21; and VWD-1C, n=8) and in AVWS-patients (AS, n=9; LVAD, n=9; and MGUS, n=8). A standard of VWF-proteolysis was specifically developed. Extent of VWF-proteolysis was expressed as relative percentage and as VWF proteolysis/VWF:Ag ratio. A speed-dependent increase in VWF-proteolysis was assessed in the LVAD model whereas no proteolysis was observed in TTP-patients. In VWD-patients, VWF-proteolysis was significantly increased in VWD-2A(IIA) and VWD-2B and significantly decreased in VWD-2A(IIE) versus controls (p< 0.0001). In AVWS-patients, VWF-proteolysis was significantly increased in AS- and LVAD-patients compared to controls (p< 0.0001) and not detectable in MGUS-patients. A significant increase in VWF-proteolysis was detected as soon as three hours after LVAD implantation (p< 0.01). In conclusion, we describe a new ELISA allowing a rapid and accurate diagnosis of VWF-proteolysis validated in three different clinical situations. This assay represents a helpful alternative to electrophoresis-based assay in the diagnosis and management of AVWS with increased VWF-proteolysis.

Transducin-like enhancer of split-1 is expressed and functional in human macrophages.

Macrophages display heterogeneous phenotypes, including the classical M1 proinflammatory and the alternative M2 anti-inflammatory polarization states. The transducin-like enhancer of split-1 (TLE1) is a transcriptional corepressor whose functions in macrophages have not been studied yet. We report that TLE1 is highly expressed in human alternative macrophages in vitro and in atherosclerotic plaques as well as in adipose tissue M1/M2 mixed macrophages. TLE1 silencing in alternative macrophages decreases the expression of the M2 markers IL-1Ra and IL-10, while it exacerbates TNFα and CCL3 induction by lipopolysaccharide. Hence, TLE1 is expressed in human macrophages where it has potential anti-inflammatory and alternative phenotype promoting properties.

M1 and M2 macrophage proteolytic and angiogenic profile analysis in atherosclerotic patients reveals a distinctive profile in type 2 diabetes.

This study aimed to investigate atherosclerotic mediators' expression levels in M1 and M2 macrophages and to focus on the influence of diabetes on M1/M2 profiles. Macrophages from 36 atherosclerotic patients (19 diabetics and 17 non-diabetics) were cultured with interleukin-1ß (IL-1ß) or IL-4 to induce M1 or M2 phenotype, respectively. The atherosclerotic mediators' expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that M1 and M2 macrophages differentially expressed mediators involved in proteolysis and angiogenesis processes. The proteolytic balance (matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/plasminogen activator inhibitor-1 (PAI-1) and MMP-9/tissue factor pathway inhibitor-2 (TFPI-2) ratios) was higher in M1 versus M2, whereas M2 macrophages presented higher angiogenesis properties (increased vascular endothelial growth factor/TFPI-2 and tissue factor/TFPI-2 ratios). Moreover, M1 macrophages from diabetics displayed more important proangiogenic and proteolytic activities than non-diabetics. This study reveals that M1 and M2 macrophages could differentially modulate major atherosclerosis-related pathological processes. Moreover, M1 macrophages from diabetics display a deleterious phenotype that could explain the higher plaque vulnerability observed in these subjects.

Successful orthotopic transplantation of short tracheal segments without immunosuppressive therapy.

OBJECTIVES: Results of tracheal transplantation have been disappointing due to of ischaemia and rejection. It has been experimentally demonstrated that results of tracheal autograft/allograft transplantation were correlated with both graft length and revascularization method. Recently, we demonstrated that heterotopic epithelium-denuded-cryopreserved tracheal allograft (TA) displayed satisfactory immune tolerance. We aimed at evaluating the results of such allografts in orthotopic transplantation according to graft length and prior heterotopic or single-stage orthotopic revascularization in a rabbit model. METHODS: Twenty New Zealand rabbits were used. Six females served as donors. Tracheal mucosa was mechanically peeled off and then the TAs were cryopreserved. Male recipients were divided into three groups receiving: (i) long TA segment with prior heterotopic revascularization (10-12 tracheal rings, n = 3); (ii) average TA segment with single-stage orthotopic revascularization (6-8 tracheal rings, n = 4); (iii) short TA segment with single-stage orthotopic revascularization (4-5 tracheal rings, n = 7). No immunosuppressive therapy was administered. Grafts were assessed bronchoscopically and upon death or sacrifice by macroscopic evaluation, histology and immunohistochemical staining for apoptosis. RESULTS: Four animals were sacrificed from Day 33 to Day 220. The survival time of other recipients was 0-47 days (mean 19.6 ± 16.7 days). Aside from three animals that died from complications, all TA segments had satisfactory stiffness, were well vascularized, showed varying levels of neoangiogenesis and inflammatory infiltration devoid of lymphocytes, and showed evidence of only low levels of apoptosis. Varying degrees of fibroblastic proliferation originating from the lamina propria were observed in the lumen of all TAs and evolved over time into collagenized fibrosis in animals surviving over 45 days. Likewise, cartilage tracheal rings exhibited central calcification deposits, which started on Day 16 and increased over time. Epithelial regeneration was constantly observed. Intense fibroblastic proliferation led to stenosis in all animals from Groups (i) and (ii) but only one of seven animals from Group (iii). CONCLUSIONS: Our results suggest that short segments of epithelium-denuded-cryopreserved TA may be reliable for tracheal transplantation in the rabbit model without problems related to graft stiffness or immune rejection. Before considering clinical applications, investigations should be conducted in larger mammals.

Immune tolerance of epithelium-denuded-cryopreserved tracheal allograft.

OBJECTIVES: Animal and clinical studies have demonstrated the feasibility of tracheal allograft transplantation after a revascularization period in heterotopy, thus requiring immunosuppressive therapy. Given the key role of the respiratory epithelium in the immune rejection, we investigated the consequence of both epithelium denudation and cryopreservation in immune tolerance of tracheal allograft in a novel rabbit model. METHODS: Five adult female New Zealand rabbits served as donors of tracheas that were denuded of their epithelium and then cryopreserved, and 13 males were used as recipients. Following graft wrap using a lateral thoracic fascial flap, allograft segments 20 mm in length with (n = 9) or without (n = 4) insertion of an endoluminal tube were implanted under the skin of the chest wall. The animals did not receive any immunosuppressive drugs. Sacrifices were scheduled up to 91 days. Macroscopic and microscopic examinations and detection of apoptotic cells by immunohistochemical staining (Apostain) were used to study the morphology, stiffness, viability and immune rejection of allografts. RESULTS: There were no postoperative complications. Grafted composite allografts displayed satisfactory tubular morphology provided that an endoluminal tube was inserted. All rabbits were found to have an effective revascularization of their allograft and a mild non-specific inflammatory infiltrate with no significant lymphocyte infiltration. Cartilage rings showed early central calcification deposit, which increased over time, ensuring graft stiffness. Apoptosis events observed into the allograft cells were suggestive of minimal chronic rejection. CONCLUSIONS: Our results demonstrated that the epithelium-denuded-cryopreserved tracheal allograft implanted in heterotopy displayed satisfactory morphology, stiffness and immune tolerance despite the absence of immunosuppressive drugs. This allograft with a fascial flap transferable to the neck should be investigated in the setting of tracheal replacement in rabbits. Similar studies need to be conducted in bigger mammals before considering clinical applications.

Liver X receptor activation stimulates iron export in human alternative macrophages.

RATIONALE: In atherosclerotic plaques, iron preferentially accumulates in macrophages where it can exert pro-oxidant activities. OBJECTIVE: The objective of this study was, first, to better characterize the iron distribution and metabolism in macrophage subpopulations in human atherosclerotic plaques and, second, to determine whether iron homeostasis is under the control of nuclear receptors, such as the liver X receptors (LXRs). METHODS AND RESULTS: Here we report that iron depots accumulate in human atherosclerotic plaque areas enriched in CD68 and mannose receptor (MR)-positive (CD68(+)MR(+)) alternative M2 macrophages. In vitro IL-4 polarization of human monocytes into M2 macrophages also resulted in a gene expression profile and phenotype favoring iron accumulation. However, M2 macrophages on iron exposure acquire a phenotype favoring iron release, through a strong increase in ferroportin expression, illustrated by a more avid oxidation of extracellular low-density lipoprotein by iron-loaded M2 macrophages. In line, in human atherosclerotic plaques, CD68(+)MR(+) macrophages accumulate oxidized lipids, which activate LXRα and LXRß, resulting in the induction of ABCA1, ABCG1, and apolipoprotein E expression. Moreover, in iron-loaded M2 macrophages, LXR activation induces nuclear factor erythroid 2-like 2 expression, thereby increasing ferroportin expression, which, together with a decrease of hepcidin mRNA levels, promotes iron export. CONCLUSIONS: These data identify a role for M2 macrophages in iron handling, a process regulated by LXR activation.

Tracheal reconstruction with a composite graft: fascial flap-wrapped allogenic aorta with external cartilage-ring support.

OBJECTIVES: Animal and clinical studies have demonstrated the feasibility of tracheal replacement by silicone-stented allogenic aortas. In clinical trials, however, this graft did not show mature cartilage regeneration into the grafts as was observed in animal models. To solve this issue, we investigated tracheal replacement with a composite graft based on a fascial flap-wrapped allogenic aorta with external cartilage-ring support in a rabbit model. METHODS: Seven male 'Géant des Flandres' and 'New Zealand' rabbits served as donors of aortas and cartilage rings, respectively. Nineteen female 'New Zealand' rabbits were used as recipients. First, in nine animals, neoangiogenesis of the composite graft following a wrap using a pedicled lateral thoracic fascial flap and implantation under the skin of the chest wall was investigated. Animal sacrifice was scheduled at regular intervals up to 38 days. Second, 10 animals underwent tracheal replacement with the composite graft after a 7-to-9 day revascularization period, and were followed-up to death. Macroscopic and microscopic examinations were used to study the morphology, stiffness and viability of the construct. RESULTS: There was one operative death after tracheal replacement. The first group of animals was found to have a satisfactory tubular morphology and stiffness of their construct associated with preserved histological structure of cartilages and moderate to severe aortic ischaemic lesions. In the group of rabbits having undergone tracheal replacement, the anatomical results were characterized by a discrepancy between the severity of ischaemic lesions involving both allogenic aorta and cartilage rings and the satisfactory biomechanical characteristics of the graft in 7 of 10 animals, probably due to cartilage calcification deposits associated with inflammatory scar tissue ensuring the stiffness of the construct. CONCLUSIONS: Our investigations demonstrate the feasibility of the replacement of circumferential tracheal defects using our composite graft. Future experiments using therapeutic bronchoscopy tools are required to draw conclusions regarding the effectiveness of this tracheal substitute in the long-term.

Clinical and prognostic implications of low or high level of von Willebrand factor in patients with Waldenstrom macroglobulinemia.

Acquired von Willebrand syndrome is described in patients with Waldenström macroglobulinemia (WM). Assessment of ristocetin cofactor activity (VWF:RCo) and von Willebrand factor (VWF) antigen (VWF:Ag) in 72 consecutive patients with WM showed a negative relation between VWF levels < 130 U/dL and both monoclonal immunoglobulin M concentration (mIgMC) and viscosity. Ten patients with VWF:RCo < 50 U/dL (< 40 for patients with blood group O) fulfilled the acquired von Willebrand syndrome criteria. They had higher mIgMC and viscosity. Reduction in mIgMC was associated with increase in VWF levels. The low VWF:RCo/VWF:Ag ratio suggested that high viscosity might be associated with increased shear force and cleavage of multimers. Surprisingly, 43 patients (59%) presented with high VWF:Ag (> 110 U/dL). They had higher bone marrow microvessel density and vascular endothelial growth factor expression on bone marrow mast cells. Five-year survival rates of patients with VWF:Ag < 110, between 110 and 250, and more than 250 U/dL were 96%, 71%, and 44%, respectively (P < .0001). High VWF:Ag was also a significant adverse prognostic factor for survival after first-line therapy (P < .0001), independently of the international scoring system. These results support systematic assessment of VWF in patients with WM. The adverse prognostic value of high VWF levels raises issues on interactions between lymphoplasmacytic cells, mast cells, and endothelial cells in WM.

Construction of a tube-shaped tracheal substitute using fascial flap-wrapped revascularized allogenic aorta.

OBJECTIVES: Animal studies have demonstrated the feasibility of tracheal replacement by silicone-stented allogenic aortas (AAs), showing mature cartilage regeneration into the grafts. In clinical trials, this graft did not prove stiff enough to allow long-term stent withdrawal. This graft insufficiency could be due to ischaemic phase prior to neoangiogenesis. To solve this issue, we investigated both the efficacy of the rabbit lateral thoracic fascial flap as a vehicle for revascularization of the AA and construction of a tube-shaped graft with transferable vascular pedicle, for more efficient replacement of the trachea. METHODS: Thirty-four New Zealand rabbits were used. After harvesting of donors 'thoracic aortas', the fresh aortic allografts were transplanted within 1 h, and the others were cryopreserved. Fifteen male and four female rabbits were used as recipients for fresh (n = 9) or cryopreserved (n = 10) aortic allografts that were implanted under the skin of the chest wall, after graft wrap using a pedicled lateral thoracic fascial flap. Animal sacrifice was scheduled at regular intervals up to 61 days. Macroscopic and microscopic examinations and fluorescence in situ hybridization (FISH) were used to study the morphology, revascularization process and viability of the construct. RESULTS: There was no operative death. Animals showed no graft rejection, despite the absence of immunosuppressive therapy. They all had a satisfactory tubular morphology of their construct. Of the 19 rabbits, 15 were found to have a generally preserved histological structure of the aorta and satisfactory neoangiogenesis. In the last four, a severe wound complication was associated with necrosis of the aortic graft. FISH on three aortic grafts with satisfactory neoangiogenesis showed migration of recipient cells into the aortic graft, decreasing from the adventitial to the luminal side, associated with the persistence of cells from the donor. CONCLUSIONS: Our results showed that the chimeric construct transformed into a well-vascularized tube-shaped organ with transferable pedicle and some degree of stiffness. Persistence of donor's cells of normal morphology into the aortic graft was suggestive of minimal ischaemia during the initial phase of revascularization. This construct might be investigated in the setting of tracheal replacement in the rabbit model.

Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways.

RATIONALE: A crucial step in atherogenesis is the infiltration of the subendothelial space of large arteries by monocytes where they differentiate into macrophages and transform into lipid-loaded foam cells. Macrophages are heterogeneous cells that adapt their response to environmental cytokines. Th1 cytokines promote monocyte differentiation into M1 macrophages, whereas Th2 cytokines trigger an "alternative" M2 phenotype. OBJECTIVE: We previously reported the presence of CD68(+) mannose receptor (MR)(+) M2 macrophages in human atherosclerotic plaques. However, the function of these plaque CD68(+)MR(+) macrophages is still unknown. METHODS AND RESULTS: Histological analysis revealed that CD68(+)MR(+) macrophages locate far from the lipid core of the plaque and contain smaller lipid droplets compared to CD68(+)MR(-) macrophages. Interleukin (IL)-4-polarized CD68(+)MR(+) macrophages display a reduced capacity to handle and efflux cellular cholesterol because of low expression levels of the nuclear receptor liver x receptor (LXR)α and its target genes, ABCA1 and apolipoprotein E, attributable to the high 15-lipoxygenase activity in CD68(+)MR(+) macrophages. By contrast, CD68(+)MR(+) macrophages highly express opsonins and receptors involved in phagocytosis, resulting in high phagocytic activity. In M2 macrophages, peroxisome proliferator-activated receptor (PPAR)γ activation enhances the phagocytic but not the cholesterol trafficking pathways. CONCLUSIONS: These data identify a distinct macrophage subpopulation with a low susceptibility to become foam cells but high phagocytic activity resulting from different regulatory activities of the PPARγ-LXRα pathways.

Atherosclerotic-like process in aortic stenosis: activation of the tissue factor-thrombin pathway and potential role through osteopontin alteration.

BACKGROUND: We recently demonstrated in an experimental model the expression of tissue factor (TF) in aortic valves. Thrombin, generated at the end of the TF-initiated coagulation cascade, has been shown to cleave the anti-calcific osteopontin (OSP) liberating the pro-inflammatory OSP N-half. OBJECTIVES: We hypothesized that TF might play an important role in calcific aortic valve stenosis (AS) through thrombin generation and hence evaluated the valvular expression of TF and its inhibitor (TF pathway inhibitor), α-thrombin, OSP and its thrombin-cleaved form (OSP N-half). METHODS: Calcified aortic valves were obtained from patients undergoing valve replacement. Protein expression was evaluated by immunostaining and measured using ELISA kits. Transcripts were analyzed by RT-PCR. RESULTS: We included 52 patients (31 men; age 70 ± 10 years; aortic valve area index 0.35 ± 0.13 cm(2)/m(2)). Immunohistochemistry revealed that TF, OSP and α-thrombin expressions were associated with calcifications at the aortic side of the leaflets. There was an overexpression in calcified regions as compared to non-calcified zones of TF (733.3 ± 70.5 pg/mg vs. 429.4 ± 73.2 pg/mg; p<0.0001), OSP (88.9 ± 12.7 ng/mg vs. 15.0 ± 3.3 ng/mg; p<0.0001) and OSP N-half (0.41 ± 0.06 pmol/mg vs. 0.056 ± 0.011 pmol/mg; p<0.0001). Additionally, both TF and α-thrombin expressions were associated with OSP N-half (r=0.52; p<0.0001 and r=0.33; p=0.019, respectively). CONCLUSIONS: Aortic leaflet TF and α-thrombin expressions and their association with the thrombin-cleaved form of OSP, are a new and important feature of AS. We hypothesize that TF may be involved in the mineralization process of aortic valves by enhancing the generation of the pro-inflammatory OSP N-half through thrombin induction. This pathway deserves further studies to address its implication in the aortic valve calcification process.

Unlike PPARgamma, PPARalpha or PPARbeta/delta activation does not promote human monocyte differentiation toward alternative macrophages.

Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an "alternative" anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPARgamma promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPARbeta/delta in this process has been reported only in mice and no data are available for PPARalpha. Here, we show that in contrast to PPARgamma, expression of PPARalpha and PPARbeta/delta overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPARgamma, PPARalpha or PPARbeta/delta activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPARalpha and PPARbeta/delta do not appear to modulate the alternative differentiation of human macrophages.

Tissue factor pathway inhibitor-2 gene methylation is associated with low expression in carotid atherosclerotic plaques.

BACKGROUND: The tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine-protease inhibitor which is expressed in atherosclerotic plaques. Epigenetic regulation of the TFPI-2 gene, through methylation of CpG islands, has been advocated in cancer. We hypothesized that TFPI-2 gene methylation could regulate TFPI-2 expression in atherosclerosis. METHODS: We used Methylation Specific PCR (MSP) and pyrosequencing in order to identify 18 CpG of the TFPI-2 promoter, in 59 carotid atherosclerotic plaques and 26 control mammary arteries. RESULTS: MSP showed methylation of the TFPI-2 gene (MSP+) in 16 plaques (27%), while no methylation (MSP-) was found in control arteries. Pyrosequencing confirmed that MSP+ plaques presented higher methylation levels than MSP- ones and arteries (p=0.03 and 0.01). Moreover, the TFPI-2 mRNA levels were lower in methylated plaques than in unmethylated ones and than in arteries (p=0.04 and <0.0001). The methylated plaques contained less lipids and macrophage infiltration than unmethylated ones. Their TFPI-2 immunoreactivity was mainly detected in the macrophages located in the media on the adventitial side, rather than in the lipid-rich core. CONCLUSION: Methylation of the TFPI-2 gene takes place in atherosclerotic plaques and is associated with decreased TFPI-2 expression. The place of this process in atherosclerosis progression remains to be investigated.

PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties.

Th1 cytokines promote monocyte differentiation into proatherogenic M1 macrophages, while Th2 cytokines lead to an "alternative" anti-inflammatory M2 macrophage phenotype. Here we show that in human atherosclerotic lesions, the expression of M2 markers and PPARgamma, a nuclear receptor controlling macrophage inflammation, correlate positively. Moreover, PPARgamma activation primes primary human monocytes into M2 differentiation, resulting in a more pronounced anti-inflammatory activity in M1 macrophages. However, PPARgamma activation does not influence M2 marker expression in resting or M1 macrophages, nor does PPARgamma agonist treatment influence the expression of M2 markers in atherosclerotic lesions, indicating that only native monocytes can be primed by PPARgamma activation to an enhanced M2 phenotype. Furthermore, PPARgamma activation significantly increases expression of the M2 marker MR in circulating peripheral blood mononuclear cells. These data demonstrate that PPARgamma activation skews human monocytes toward an anti-inflammatory M2 phenotype.

Decellularized heart valve as a scaffold for in vivo recellularization: deleterious effects of granulocyte colony-stimulating factor.

BACKGROUND: Autologous recellularization of decellularized heart valve scaffolds is a promising challenge in the field of tissue-engineered heart valves and could be boosted by bone marrow progenitor cell mobilization. The aim of this study was to examine the spontaneous in vivo recolonization potential of xenogeneic decellularized heart valves in a lamb model and the effects of granulocyte colony-stimulating factor mobilization of bone marrow cells on this process. METHODS: Decellularized porcine aortic valves were implanted in 12 lambs. Six lambs received granulocyte colony-stimulating factor (10 microg x kg(-1) x d(-1) for 7 days, granulocyte colony-stimulating factor group), and 6 received no granulocyte colony-stimulating factor (control group). Additionally, nondecellularized porcine valves were implanted in 5 lambs (xenograft group). Angiographic and histologic evaluation was performed at 3, 6, 8, and 16 weeks. RESULTS: Few macroscopic modifications of leaflets and the aortic wall were observed in the control group, whereas progressive shrinkage and thickening of the leaflets appeared in the granulocyte colony-stimulating factor and xenograft groups. In the 3 groups progressive ovine cell infiltration (fluorescence in situ hybridization) was observed in the leaflets and in the adventitia and the intima of the aortic wall but not in the media. Neointimal proliferation of alpha-actin-positive cells, inflammatory infiltration, adventitial neovascularization, and calcifications were more important in the xenograft and the granulocyte colony-stimulating factor groups than in the control group. Continuous re-endothelialization appeared only in the control group. CONCLUSION: Decellularized xenogeneic heart valve scaffolds allowed partial autologous recellularization. Granulocyte colony-stimulating factor led to accelerated heart valve deterioration similar to that observed in nondecellularized xenogeneic cardiac bioprostheses.

Dietary lipid lowering modifies plaque phenotype in rabbit atheroma after angioplasty: a potential role of tissue factor.

BACKGROUND: Tissue factor (TF), the main initiator of blood coagulation, is involved in cellular migration and angiogenesis processes. TF is expressed strongly in lipid-rich plaques and probably plays an important role in the thrombotic complications of plaque rupture. This study analyzes the effect of dietary lipid lowering on TF expression and cellular modifications in angioplasty-induced rabbit plaque rupture. METHODS AND RESULTS: After experimental plaque rupture by balloon angioplasty in atheromatous rabbits, animals were assigned a 0.2% or a 2% cholesterol diet, and the TF content of arterial wall and the associated histological modifications were analyzed after 4 weeks. Early effects of lipid lowering were observed: The increase of TF expression in the vascular wall was stronger in the 2% than in the 0.2% cholesterol diet group (iliac arteries: 1226+/-308 versus 72+/-29 mU TF/g artery, P<0.005). Immunohistochemistry indicated that TF expression was associated with sprout of neovessels, which was more pronounced in the 2% than in the 0.2% cholesterol group. CONCLUSIONS: This study shows that dietary lipid lowering decreases the thrombotic potential of ruptured atherosclerotic plaques through TF decrease. Moreover, high TF expression is associated with marked angiogenesis in the vascular wall, which is reduced by lipid lowering. These results provide further arguments for strong dietary lipid lowering to reduce plaque instability and thrombogenicity.