Impact of the neutrophil response to granulocyte colony-stimulating factor on the risk of hemorrhage when used in combination with tissue plasminogen activator during the acute phase of experimental stroke.

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF) is a pharmacologic agent inducing neutrophil mobilization and a new candidate for neuroprotection and neuroregeneration in stroke. Its effects when used in combination with tissue plasminogen activator (tPA) were explored during the acute phase of ischemic stroke. METHODS: We used a middle cerebral artery occlusion (MCAO) model of cerebral ischemia, associated with treatment with tPA, in male spontaneously hypertensive rats (SHR). Granulocyte colony-stimulating factor (G-CSF; 60 µg/kg) was injected just before tPA. Neutrophil response in peripheral blood and in the infarct area was quantified in parallel to the infarct volume. Protease matrix metallopeptidase 9 (MMP-9) release from circulating neutrophils was analyzed by immunochemistry and zymography. Vascular reactivity and hemorrhagic volume in the infarct area was also assessed. RESULTS: Twenty four hours after ischemia and tPA, G-CSF administration induced a significant increase of neutrophils in peripheral blood (P <0.05). At 72 hours post-ischemia, G-CSF was significantly associated with an increased risk of hemorrhage in the infarct area (2.5 times more likely; P <0.05) and significant cerebral endothelium-dependent dysfunction. Ex vivo, an increased MMP-9 release from neutrophils after tPA administration correlated to the increased hemorrhagic risk (P <0.05). In parallel, G-CSF administration was associated with a decreased neutrophil infiltration in the infarct area (-50%; P <0.05), with a concomitant significant neuroprotective effect (infarct volume: -40%; P <0.05). CONCLUSIONS: We demonstrate that G-CSF potentiates the risk of hemorrhage in experimental stroke when used in combination with tPA by inducing neutrophilia. This effect is concomitant to an increased MMP-9 release from peripheral neutrophils induced by the tPA treatment. These results highlight the potential hemorrhagic risk of associating G-CSF to thrombolysis during the acute phase of stroke.

Microbiota-sensitive drug delivery systems based on natural polysaccharides for colon targeting.

Colon targeting is an ongoing challenge, particularly for the oral administration of biological drugs or local treatment of inflammatory bowel disease (IBD). In both cases, drugs are known to be sensitive to the harsh conditions of the upper gastrointestinal tract (GIT) and, thus, must be protected. Here, we provide an overview of recently developed colonic site-specific drug delivery systems based on microbiota sensitivity of natural polysaccharides. Polysaccharides act as a substrate for enzymes secreted by the microbiota located in the distal part of GIT. The dosage form is adapted to the pathophysiology of the patient and, thus, a combination of bacteria-sensitive and time-controlled release or pH-dependent systems can be used for delivery.

Modulation of inflammatory M1-macrophages phenotype by valvular interstitial cells.

BACKGROUND: Aortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. METHODS: We first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. RESULTS: Circulating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell-conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell-conditioned media from control aortic valve donors. CONCLUSIONS: These findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.

Analysis of the association of MPO and MMP-9 with stroke severity and outcome: Cohort study.

OBJECTIVE: In acute cerebral ischemia, circulating neutrophil count and neutrophil-to-lymphocyte ratio (NLR) are positively associated with stroke severity and worse outcomes. Mediators of this effect are unknown. We aimed to investigate (1) the relationship between plasma matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) concentrations with stroke severity and outcome and (2) MMP-9 and MPO release after ex vivo stimulation of neutrophils by recombinant tissue plasminogen activator (rtPA). METHODS: We analyzed data collected in 255 patients with supratentorial cerebral infarcts recruited within 48 hours of symptoms onset irrespective of rtPA treatment. The endpoints were excellent outcome (modified Rankin Scale score 0-1), symptomatic intracerebral hemorrhage (European Cooperative Acute Stroke Study-II definition), and death at 3 months. The role of rtPA treatment on peripheral neutrophil degranulation was investigated in 18 patients within 4.5 hours and after 72 hours. RESULTS: Neutrophil counts, NLR, and MPO plasma concentrations, but not MMP-9, were positively correlated with stroke severity. Higher neutrophil counts and NLR were independently associated with worse outcomes and higher mortality rates at month 3. Higher MPO plasma concentrations, but not MMP-9, were associated with worse outcome. Neutrophil-derived MMP-9, after ex vivo rtPA stimulation, but not MPO, were higher after 72 hours in patients treated by IV rtPA but not associated with hemorrhagic transformation. CONCLUSIONS: Neutrophil counts, NLR, and MPO plasma concentrations are associated with worse outcome in patients with acute cerebral ischemia, in contrast to MMP-9. Further investigations are needed to deepen our knowledge on MPO's role in the deleterious effect of neutrophils because it could represent a potential therapeutic target.

The tumour suppressor CDKN2A/p16INK4a regulates adipogenesis and bone marrow-dependent development of perivascular adipose tissue.

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Leptin induces osteoblast differentiation of human valvular interstitial cells via the Akt and ERK pathways.

AIMS: Calcific aortic valve disease (CAVD) affects 2-6% of the population over 65 years, and age, gender, smoking, overweight, dyslipidemia, diabetes contribute to the development of this disease. CAVD results, in part, from the osteoblast differentiation of human valvular interstitial cells (VICs). This study aims to elucidate the effects of leptin on osteoblast phenotype of VICs and the signalling pathways involved. METHODS: Patients who underwent aortic valve replacement for CAVD (n = 43) were included in this study. Patients with coronary artery disease (CAD) without CAVD (n = 129) were used as controls. RESULTS: Patients with CAVD had higher serum leptin concentrations than CAD patients (p = 0.002). Leptin was found in calcific aortic valves, with higher concentrations in calcified versus non-calcified zones (p = 0.01). Chronic leptin stimulation of human VICs enhanced alkaline phosphatase (ALP) activity and ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. Moreover, inhibiting Akt or ERK during leptin stimulation lowered the expression of osteoblast markers in VIC. CONCLUSIONS: Taken together, these findings indicate that leptin plays a critical role in CAVD development by promoting osteoblast differentiation of human aortic VICs in an Akt- and ERK-dependent manner. This study highlights the role of leptin in CAVD development, and further studies are needed to determine whether reducing circulating leptin levels or blocking leptin actions on VICs is efficient to slow CAVD progression.

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.

Alterations in neutrophil production and function at an early stage in the high-fructose rat model of metabolic syndrome.

BACKGROUND: Although neutrophils are crucially involved in inflammation, they have received only little attention in metabolic syndrome (MetS). We hypothesized that neutrophil infiltration into adipose tissue (AT) may occur at an early stage of MetS, in association with modulation of major functions of neutrophils and of their bone marrow production. METHODS: Fifty-six male Sprague-Dawley rats were fed regular (control rats (CRs)) or high-fructose (60%; fructose-fed rats (FFRs)) diets. After 6 weeks, metabolic parameters were measured. Distribution of neutrophils into AT was investigated by immunohistochemistry. Function of circulating neutrophils (activation, reactive oxygen species production, phagocytosis, and apoptosis) was determined by flow cytometry. Granulopoiesis was evaluated by measuring the number and survival characteristics of neutrophil progenitors using bone marrow culture assays and flow cytometry. RESULTS: Compared with the CR group, the FFR group developed MetS (i.e., arterial hypertension, hypertriglyceridemia, fasting hyperglycemia, and greater intra-abdominal AT volume) and presented higher neutrophil infiltration into AT. At resting state, no significant difference for circulating neutrophil functions was observed between the 2 groups. In contrast, circulating neutrophils from the FFR group exhibited higher responses to phorbol-12-myristate-13-acetate for all studied functions, compared with the CR group, suggesting that early MetS induces neutrophil priming. In parallel, a diminished clonal capacity and an increased apoptosis in bone marrow-derived granulocyte progenitors and neutrophil precursors were observed in the FFR group compared with the CR group. CONCLUSIONS: These results provide evidence of an increased infiltration into intra-abdominal AT and modified production, function, and phenotype of neutrophils at an early stage of high-fructose diet-induced MetS.

Frequency of abdominal aortic aneurysm in patients undergoing coronary artery bypass grafting.

The aims of this study were to clarify the prevalence and the risk factors for unsuspected abdominal aortic aneurysm (AAA) in patients who underwent coronary artery bypass grafting for severe coronary artery disease and to identify the most at risk patients for AAA. Among 217 patients (189 men, mean age 64 +/- 11 years), asymptomatic AAAs, as prospectively identified by echocardiography, were found in 15 patients (6.9%). All patients with AAAs were men and smokers or past smokers. Factors significantly associated by univariate analysis with asymptomatic AAA presence were smoking (p = 0.003), symptomatic peripheral artery disease (p = 0.006), significant carotid artery stenosis (p = 0.007), and larger femoral and popliteal diameters (p = 0.008 and p = 0.0012, respectively). The other classic demographic, clinical, and biologic features were equally distributed among patients. In conclusion, in patients who underwent coronary artery bypass grafting who were men and aged <75 years with smoking histories, the prevalence of AAA was as high as 24% when they had concomitant peripheral arterial disease and/or carotid artery stenosis (vs 4.4% in the absence of either condition, p = 0.007), justifying consideration of AAA screening in this subgroup of in-hospital patients.

Characterization of a homozygous Gly11Val mutation in the Gla domain of coagulation factor X.

Factor (F) X deficiency is a rare inherited autosomal recessive trait. We report on a patient affected by a severe bleeding diathesis. Mutations were sought by F10 sequence analysis. The consequences of the mutation were characterized by measuring thrombin and FXa formation after triggering the clotting cascade with activated partial thromboplastin time (aPTT) reagent or with phospholipid vesicles plus either tissue factor (TF) or FIXabeta, or with the FX activator from Russell's viper venom (RVV-X). The patient was found to be homozygous for a novel FX p.G51V mutation (G11V of the mature protein) within the omega-loop of the gamma-carboxyglutamic-rich domain. FX activity was markedly reduced (FX:C <1%) in prothrombin time and aPTT assays, and was 15% of normal in the RVV-X assay. The antigen level (FX:Ag) was 75%. TF, alone or in combination with recombinant FVIIa, failed to trigger detectable FXa or thrombin activity in the patient's plasma. FIXabeta also failed to trigger measurable FXa or thrombin production, but activation with RVV-X was only 4-fold less effective in the patient's plasma than in normal plasma. Supplementation with normal FX suggested that FX(G11V) and/or FXa(G11V) might slow the clotting cascade by competition. Overall, the patient's phenotype appears to be due to a very low rate of FX(G11V) activation by TF/FVIIa and FVIIIa/FIXa complexes rather than to FXa(G11V) activity within prothrombinase.

Platelet factor 4 (CXCL4) seals blood clots by altering the structure of fibrin.

Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31- and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 microm the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.

Thrombin-activable factor X re-establishes an intrinsic amplification in tenase-deficient plasmas.

Classical hemophilia results from a defect of the intrinsic tenase complex, the main factor X (FX) activator. Binding of factor VIIa to tissue factor triggers coagulation, but little amplification of thrombin production occurs. Handling of hemophilia by injection of the deficient or missing (thus foreign) factor often causes immunological complications. Several strategies have been designed to bypass intrinsic tenase complex, but none induce true auto-amplification of thrombin production. In an attempt to re-establish a cyclic amplification of prothrombin activation in the absence of tenase, we prepared a chimera of FX having fibrinopeptide A for the activation domain (FX(FpA)). We reasoned that cascade initiation would produce traces of thrombin that would activate FX(FpA) (contrary to its normal homologue). Given that the activation domain of FX is released upon activation, thrombin cleavage would produce authentic FXa that would produce more thrombin, which in turn would activate more chimeras. FX(FpA) was indeed activable by thrombin, albeit at a relatively low rate (5 x 10(3) M(-1) s(-1)). Nevertheless, FX(FpA) allowed in vitro amplification of thrombin production, and 100 nM efficiently corrected thrombin generation in tenase-deficient plasmas. A decisive advantage of FX(FpA) could be that the artificial cascade is self-regulating: FX(FpA) had little influence on the clotting time of normal plasma, yet corrected that of tenase deficiency. Another advantage could be the half-life of FX(FpA) in blood; FX has a half-life of about 30 h (less than 3 h for FVIIa). It is also reasonable to expect little or no immunogenicity, because FX and fibrinopeptide A both circulate normally in the blood of hemophiliacs.