International audienceIntravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that hemeladen MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of hemeladen MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

Patients with sickle cell disease suffer from painful crises associated with disseminated vaso-occlusions, increased circulating erythrocyte microparticles (MPs), and thrombospondin-1 (TSP1). MPs are submicron membrane vesicles shed by compromised or activated cells. We hypothesized that TSP1 mediates MP shedding and participates in vaso-occlusions. We injected TSP1 to transgenic SAD mice with sickle cell disease and characterized circulating phosphatidylserine؉ MPs by FACS. TSP1 stimulated MPs in plasma and initiated vaso-occlusions within minutes. In vitro, TSP1 triggered rapid erythrocyte conversion into spicule-covered echinocytes, followed by MP shedding. MP shedding was recapitulated by peptides derived from the TSP1 carboxyterminus. We purified MPs shed by erythrocytes in vitro and administered them back to SAD mice. MPs triggered immediate renal vaso-occlusions. In vitro, MPs triggered the production of radical oxygen species by endothelial monolayers, favored erythrocyte adhesion, and induced endothelial apoptosis. MPs also compromised vasodilation in perfused microvessels. These effects were inhibited by saturating MP phosphatidylserine with annexin-V, or with inhibitors of endothelial ROS production. We conclude that TSP1 triggers erythrocyte MP shedding. These MPs induce endothelial injury and facilitate acute vaso-occlusive events in transgenic SAD mice. This work supports a novel concept that toxic erythrocyte MPs may connect sickle cell anemia to vascular disease. (Blood. 2012;120(25): 5050-5058) IntroductionSickle cell disease (SCD) is a form of hemolytic anemia that results from a point mutation (HbS) in the gene of the globin ␤-chain. The mutation makes HbS susceptible to polymerize during hypoxic stress, producing long intracellular fibers that are responsible for drastic membrane and cytoskeletal remodeling. The HbS fibers polymerize and depolymerize in response to blood oxygenation and deoxygenation cycles. The repeated changes in red blood cell (RBC) shape cause a permanent loss of the biconcave phenotype, producing condensed cells with diverse degrees of crenation, and covered in cone-shaped membrane figures known as spicules. [1][2][3] The remodeled erythrocytes become progressively dehydrated and rigid. Fully and irreversibly remodeled erythrocytes are termed sickle erythrocytes with respect to their ultimate shape.Sickle erythrocytes display a strong predisposition to adhere to the endothelium, get trapped in small capillaries and reduce blood flow. Highly vascularized organs, such as kidneys, bones, and lungs are the seat of disseminated vascular occlusions and recurrent ischemic injury. 4 In some of these organs, ischemia causes extremely painful episodes termed vaso-occlusive crises (VOCs). These crises are attributed to stress factors, such as hypoxia, physical exercise, infection, trauma, or stress. However, the molecular basis for the development of vaso-occlusions in SCD has yet to be explained and several different triggers of VOCs may exist.After getting trapped in capillaries,...

Lactadherin is a secreted extracellular matrix protein expressed in phagocytes and contributes to the removal of apoptotic cells. We examined lactadherin expression in brain sections of patients with or without Alzheimer's disease and studied its role in the phagocytosis of amyloid ␤-peptide (A␤). Cells involved in Alzheimer's disease, including vascular smooth muscle cells, astrocytes, and microglia, showed a time-related increase in lactadherin production in culture. Quantitative analysis of the level of lactadherin showed a 35% reduction in lactadherin mRNA expression in the brains of patients with Alzheimer's disease (n ‫؍‬ 52) compared with age-matched controls (n ‫؍‬ 58; P ‫؍‬ 0.003). Interestingly, lactadherin protein was detected in the brains of patients with Alzheimer's disease and controls, with low expression in areas rich in senile plaques and marked expression in areas without A␤ deposition. Using surface plasmon resonance, we observed a direct pro-

We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4+CD8dim+ and CD4+CD8− phenotype. Both clones mediated a specific MHC class I–restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vβ gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vβ7/Jβ2.3, Vβ13/Jβ2.5, and Vβ22/Jβ2.5 rearrangements. Phenotypic analysis using specific anti-Vβ monoclonal antibodies indicated that only Vβ13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vβ gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vβ transcript, corresponding, respectively, to Vβ5/Jβ2.3 and Vβ17/Jβ2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vβ TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.

Photodynamic therapy is an emerging cancer treatment that is particularly adapted for localized malignant tumor. The phototherapeutic agent is generally injected in the bloodstream and circulates in the whole organism as a chemotherapeutic agent, but needs light triggering to induce localized therapeutic effects. We found that one of the responses of in vitro and in vivo cancer cells to photodynamic therapy was a massive production and emission of extracellular vesicles (EVs): only 1 hour after the photo-activation, thousands of vesicles per cell were emitted in the extracellular medium. A similar effect has been found after treatment with Doxorubicin (chemotherapy), but far less EVs were produced, even 24 hours after the treatment. Furthermore, we found that the released EVs could transfer extracellular membrane components, drugs and even large intracellular objects to naive target cells. In vivo, photodynamic treatment and chemotherapy increased the levels of circulating EVs several fold, confirming the vast induction of cancer cell vesiculation triggered by anti-cancer therapies.

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Background-Defects in cardiomyocyte Ca2ϩ cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca 2ϩ cycling, but the potential relevance of this in HF is unknown. Methods and Results-We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca 2ϩ handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (PϽ0.0001 versus controlϩTAC) and less left ventricular dilatation than controlϩTAC mice (PϽ0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7Ϯ0.6% versus 30.3Ϯ2.6% in controlϩTAC mice, PϽ0.05). Isolated cardiomyocytes from DTϩTAC mice had greater shortening, intracellular Ca 2ϩ transients, and sarcoplasmic reticulum Ca 2ϩ load (PϽ0.05 versus controlϩTAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status. Conclusions-Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressureoverload-induced HF through modulation of calcium cycling. (Circulation. 2008;117:3187-3198.)