CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model.

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs.

Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events.

Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.

Anucleate platelets generate progeny.

Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and alpha-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signal-dependent expression of surface P-selectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream.

PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids.

Platelet activating factor (PAF) and PAF-like lipids induce inflammatory responses in target cells. These lipid mediators are inactivated by PAF-acetylhydrolase (PAF-AH). The PAF signaling system affects the growth of hematopoietic CD34(+) cells, but roles for PAF-AH in this process are unknown. Here, we investigated PAF-AH function during megakaryopoiesis and found that human CD34(+) cells accumulate this enzymatic activity as they differentiate toward megakaryocytes, consistent with the expression of mRNA and protein for the plasma PAF-AH isoform. Inhibition of endogenous PAF-AH activity in differentiated megakaryocytes increased formation of lipid mediators that signaled the PAF receptor (PAFR) in fully differentiated human cells such as neutrophils, as well as megakaryocytes themselves. PAF-AH also controlled megakaryocyte alpha(IIb)beta(3)-dependent adhesion, cell spreading, and mobility that relied on signaling through the PAFR. Together these data suggest that megakaryocytes generate PAF-AH to modulate the accumulation of intracellular phospholipid mediators that may detrimentally affect megakaryocyte development and function.

Megakaryocyte impairment by eptifibatide-induced antibodies causes prolonged thrombocytopenia.

Glycoprotein (GP) IIbIIIa inhibitors are used in the treatment of acute coronary syndromes. Transient immune-mediated acute thrombocytopenia is a recognized side effect of GPIIbIIIa inhibitors. We provide evidence that GPIIbIIIa inhibitor-induced antibodies can affect megakaryocytes in the presence of eptifibatide. In a patient with acute coronary syndrome, acute thrombocytopenia occurred after a second exposure to eptifibatide 20 days after the initial treatment. Despite the short half-life of eptifibatide (t(1/2) = 2 hours), thrombocytopenia less than 5 x 10(9)/L and gastrointestinal and skin hemorrhage persisted for 4 days. Glycoprotein-specific enzyme-linked immunosorbent assay showed eptifibatide-dependent, GPIIbIIIa-specific antibodies. Bone marrow examination showed predominance of early megakaryocyte stages, and platelet transfusion resulted in an abrupt platelet count increase. Viability of cultured cord blood-derived megakaryocytes was reduced in the presence of eptifibatide and patient IgG fraction. These findings can be explained by impaired megakaryocytopoiesis complicating anti-GPIIbIIIa antibody-mediated immune thrombocytopenia. This mechanism may also apply to some patients with autoimmune thrombocytopenia.

Association of low-grade inflammation and platelet activation in patients with hypertension with microalbuminuria.

Increased levels of sCD40L (soluble CD40 ligand) have been associated with enhanced in vivo platelet activation, and may represent a molecular link between inflammation and a prothrombotic state. The aim of the present study was to analyse the relationship between platelet activation, endothelial dysfunction, low-grade inflammation and sCD40L in patients with hypertension with or without MA (microalbuminuria). A cross-sectional comparison of sCD40L levels was performed in 25 patients with MH (essential hypertension with MA) pair-matched for gender and age with 25 patients with EH (essential hypertension) and 25 HS (healthy subjects with normotension). Circulating levels of CRP (C-reactive protein), a marker of inflammation, sP-selectin (soluble P-selectin), a marker of in vivo platelet activation, and ADMA (asymmetric dimethylarginine) and vWF (von Willebrand factor), markers of endothelial dysfunction, were analysed in each subject. sCD40L levels were increased in patients with MH compared with either patients with EH (P<0.001) or HS (P<0.0001). A highly significant correlation between plasma sCD40L and sP-selectin (P<0.0001), vWF (P<0.001) or CRP levels (P<0.05) was observed in patients with MH. Multivariate regression analysis showed that sP-selectin was the strongest independent predictor of sCD40L levels (P<0.0001) in patients with MH. Patients with hypertension with both vWF and CRP levels above the median had the highest sCD40L levels (P<0.0001). Factorial ANOVA of all of the patients with hypertension confirmed that only patients with MH with low-grade inflammation had elevated levels of sCD40L. In conclusion, sCD40L levels appear to discriminate a subset of patients characterized by MA and low-grade inflammation, suggesting that inhibition of the CD40/CD40L system may represent a potential therapeutic target in subjects with hypertension at a high risk of cardiovascular events.

Soluble RAGE in type 2 diabetes: association with oxidative stress.

Advanced glycation end products (AGEs) contribute to diabetic vascular complications by engaging the AGE receptor (RAGE). A soluble RAGE form (sRAGE) acts as a decoy domain receptor, thus decreasing AGE cellular binding. A cross-sectional comparison of sRAGE, asymmetric dimethylarginine (ADMA) plasma levels (index of endothelial dysfunction), and urinary 8-iso-prostaglandin (PG)F(2alpha) (marker of oxidative stress) was performed between 86 diabetic patients and 43 controls. Plasma sRAGE levels were significantly lower and ADMA levels were significantly higher in diabetic patients as compared to controls (P<0.0001). HbA1c and urinary 8-iso-PGF(2alpha) were correlated inversely with sRAGE and directly with ADMA. On multivariate analysis HbA1c was independently related to sRAGE levels in diabetic patients. Twenty-four of 86 patients with newly diagnosed diabetes and 12 patients in poor metabolic control were reevaluated after treatment with a hypoglycemic agent or insulin, respectively. Improvement in metabolic control by oral agents or insulin resulted in a significant increase in sRAGE and decrease in ADMA levels (P<0.0001). Thus, poor glycemic control reduces sRAGE levels, in association with enhanced oxidative stress and endothelial dysfunction in diabetes. These abnormalities are susceptible to modulation by improvement in metabolic control.

Decreased in vivo oxidative stress and decreased platelet activation following metformin treatment in newly diagnosed type 2 diabetic subjects.

BACKGROUND: In type 2 diabetes, metformin reduces cardiovascular risk beyond the effect of glycaemic control. Since oxidative stress and the consequent enhanced platelet activation contribute to accelerated atherosclerosis in diabetes, we hypothesized that metformin could reduce oxidative stress in this condition. METHODS: We randomized 26 newly diagnosed type 2 diabetic subjects to assume either metformin (M, n = 13) or gliclazide (G, n = 13) for 12 weeks. Drugs were titrated as needed to achieve good glycaemic control. Before and after treatment, we determined blood glucose, insulin, HbA(1c), vitamin A and E levels and 8-iso-PGF(2alpha) and 11-dehydro-thromboxane B(2) urinary excretion, an in vivo oxidative stress and a thromboxane-dependent platelet activation marker, respectively. RESULTS: Notwithstanding a comparable improvement in metabolic control, 8-iso-PGF(2alpha) (M from 708 +/- 32 to 589 +/- 45 pg/mg cr, p < 0.001; G from 646 +/- 80 to 665 +/- 79, pg/mg cr, p = ns) and 11-dehydro-thromboxane B(2) (M from 2190 +/- 196 to 1753 +/- 150 pg/mg cr, p < 0.05; G from 2048 +/- 202 to 1923 +/- 223, pg/mg cr, p = ns) urinary excretion decreased after metformin but not after gliclazide treatment. After metformin, vitamin A and E levels significantly increased while they remained unchanged after gliclazide. CONCLUSIONS: These data suggest that metformin could improve oxidative stress, preserve antioxidant function and restrain platelet activation in type 2 diabetes.