Low grade intravascular hemolysis associates with peripheral nerve injury in type 2 diabetes.

Type 2 diabetes (T2D) induces hyperglycemia, alters hemoglobin (Hb), red blood cell (RBC) deformability and impairs hemorheology. The question remains whether RBC breakdown and intravascular hemolysis (IVH) occur in T2D patients. We characterized RBC-degradation products and vesiculation in a case-control study of 109 T2D patients and 65 control subjects. We quantified heme-related absorbance by spectrophotometry and circulating extracellular vesicles (EV) by flow cytometry and electron microscopy. Heme-related absorbance was increased in T2D vs. control plasma (+57%) and further elevated in obese T2D plasma (+27%). However, large CD235a+ EV were not increased in T2D plasma. EV from T2D plasma, or shed by isolated T2D RBC, were notably smaller in diameter (-27%) and carried heme-related absorbance. In T2D plasma, higher heme-related absorbance (+30%) was associated to peripheral sensory neuropathy, and no other vascular complication. In vitro, T2D RBC-derived EV triggered endothelial stress and thrombin activation in a phosphatidylserine- and heme-dependent fashion. We concluded that T2D was associated with low-grade IVH. Plasma absorbance may constitute a novel biomarker of peripheral neuropathy in T2D, while flow cytometry focusing on large EV may be maladapted to characterize RBC EV in T2D. Moreover, therapeutics limiting IVH or neutralizing RBC breakdown products might bolster vasculoprotection in T2D.

Exploring the microvascular impact of red blood cell transfusion in intensive care unit patients.

BACKGROUND: Red blood cell (RBC) transfusion is a common treatment for hospitalized patients. However, the effects of RBC transfusion on microvascular function remain controversial. METHODS: In a medical ICU in a tertiary teaching hospital, we prospectively included anemic patients requiring RBC transfusion. Skin microvascular reactivity was measured before and 30 min after RBC transfusion. Plasma was collected to analyze intravascular hemolysis and draw the lipidomic and cytokine profiles. RESULTS: In a cohort of 59 patients, the median age was 66 [55-81] years and SAPS II was 38 [24-48]. After RBC transfusion, endothelium-dependent microvascular reactivity improved in 35 (59%) patients, but worsened in 24 others (41%). Comparing clinical and biological markers revealed that baseline blood leucokyte counts distinguished improving from worsening patients (10.3 [5.7; 19.7] vs. 4.6 [2.1; 7.3] × 109/L; p = 0.001) and correlated with variations of microvascular reactivity (r = 0.36, p = 0.005). Blood platelet count was also higher in improving patients (200 [97; 280] vs 160 [40; 199] × 103/mL, p = 0.03) but did not correlate with variations of microvascular reactivity. We observed no intravascular hemolysis (HbCO, heme, bilirubin, LDH), but recorded a significant increase in RBC microparticle levels specific to improving patients after transfusion (292 [108; 531] vs. 53 [34; 99] MP/µL; p = 0.03). The improvement in microvascular dilation was positively correlated with RBC microparticle levels (R = 0.83, p < 0.001) and conversion of arachidonic acid into vasodilating eicosanoids. CONCLUSIONS: Patients displaying an improved microvascular reactivity after RBC transfusion had high blood leukocyte counts, increased RBC microparticle formation, and enhanced metabolism of arachidonic acid into vasodilating lipids. Our data suggested a contribution of recipient leukocytes to the vascular impact of RBC transfusion.

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

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.

Massive release of extracellular vesicles from cancer cells after photodynamic treatment or chemotherapy.

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.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

Intravascular 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 heme-laden 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 heme-laden 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.

Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease.

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.

C/EBP homologous protein-10 (CHOP-10) limits postnatal neovascularization through control of endothelial nitric oxide synthase gene expression.

BACKGROUND: C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. METHODS AND RESULTS: Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10(-/-) mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10(-/-) mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10(-/-) mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA-transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10(-/-) mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. CONCLUSIONS: This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.

Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling.

BACKGROUND: Defects in cardiomyocyte Ca(2+) 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(2+) 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(2+) 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 (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7+/-0.6% versus 30.3+/-2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load (P<0.05 versus control+TAC 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 pressure-overload-induced HF through modulation of calcium cycling.

Evidence of a role for lactadherin in Alzheimer's disease.

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 beta-peptide (Abeta). 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 Abeta deposition. Using surface plasmon resonance, we observed a direct protein-protein interaction between recombinant lactadherin and Abeta 1-42 peptide in vitro. Lactadherin deficiency or its neutralization using specific antibodies significantly prevented Abeta 1-42 phagocytosis by murine and human macrophages. In conclusion, lactadherin plays an important role in the phagocytosis of Abeta 1-42 peptide, and its expression is reduced in Alzheimer's disease. Alterations in lactadherin production/function may contribute to the initiation and/or progression of Alzheimer's disease.

Association of annexin A5 with Na+/Ca2+ exchanger and caveolin-3 in non-failing and failing human heart.

Annexin A5 is a Ca2+ dependent phosphatidylserine binding protein mainly located in the T-tubules and sarcolemma of cardiomyocytes. Our objectives were to determine whether annexin A5 was associated with various protein(s) and whether such an association was modified in failing (F) hearts. The association between annexin A5 and the cardiac Na+/Ca2+ exchanger (NCX) was demonstrated by immunohistofluorescence, annexin A5-biotin overlay and co-immunoprecipitations (IPs) performed with microsomal preparations (MPs) from non-failing (NF) (n = 8) and F (dilated cardiomyopathy, n = 7) human hearts. We moreover found caveolin-3 in the immunoprecipitates, indicating the presence of multimolecular subsarcolemmal complexes. Surface plasmon resonance assays in NF MPs allowed us to demonstrate direct interaction between the NCX and caveolin-3 and immobilized annexin A5. Interaction was Ca2+-dependent and inhibited by the specific antibody. In addition, dissociation by zwittergent 3-14 (ZW 3-14) of the complexes from MPs increased specific interactions. In F hearts, specific interactions were blunted in native MPs but were fully recovered after treatment with ZW 3-14. In conclusion, we demonstrated that a direct interaction between annexin A5 and the cardiac NCX occurs in complexes including caveolin-3. In F hearts, despite the increase in the exchanger level, almost all of the NCX was involved in complexes. These interactions probably occurred in the intracytoplasmic regulatory loop of the exchanger, suggesting a different regulation of the exchanger in heart failure, consistent with a role in altered Ca2+ handling.

Externalization of endogenous annexin A5 participates in apoptosis of rat cardiomyocytes.

OBJECTIVE: Annexins are Ca(2+)-dependent phospholipid binding proteins. Externalized annexin A5 has been recently suggested to have a proapoptotic effect. Our aim was to determine whether annexin A5, which is intracellular in cardiomyocytes, could be translocated and/or externalized and play a role during the apoptotic process. METHODS: Apoptosis was induced in rat cardiomyocytes by continuous incubation with staurosporine or 30 min treatment with H(2)O(2) and was measured by phosphatidylserine (PS) externalization, TUNEL staining and DNA ladder. Immunofluorescence labeling of annexin A5 was performed on permeabilized or nonpermeabilized cardiomyocytes. RESULTS: Staurosporine or H(2)O(2) treatment of neonatal cardiomyocytes resulted in significant increases of apoptosis at 24 h, but H(2)O(2) treatment led to a faster and higher PS externalization than that observed with ST. In both neonatal and adult cardiomyocytes, annexin A5 was intracellular in control conditions but was found at the external face of sarcolemma during apoptosis. Furthermore, neonatal cardiomyocytes with externalized annexin A5 have apoptotic characteristics and their number increased with time. Interestingly, immediately after H(2)O(2) induction, the number of annexin A5-positive cells was higher than that of PS-positive cells (p