Hydroxychloroquine inhibits hemolysis-induced arterial thrombosis ex vivo and improves lung perfusion in hemin-treated mice.

BACKGROUND: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis. OBJECTIVES: To investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis. METHODS: The effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl3)-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice. RESULTS: Erythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride-induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi. CONCLUSION: Hydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion.

Modelling the pathology and treatment of cardiac fibrosis in vascularised atrial and ventricular cardiac microtissues.

Introduction: Recent advances in human cardiac 3D approaches have yielded progressively more complex and physiologically relevant culture systems. However, their application in the study of complex pathological processes, such as inflammation and fibrosis, and their utility as models for drug development have been thus far limited. Methods: In this work, we report the development of chamber-specific, vascularised human induced pluripotent stem cell-derived cardiac microtissues, which allow for the multi-parametric assessment of cardiac fibrosis. Results: We demonstrate the generation of a robust vascular system in the microtissues composed of endothelial cells, fibroblasts and atrial or ventricular cardiomyocytes that exhibit gene expression signatures, architectural, and electrophysiological resemblance to in vivo-derived anatomical cardiac tissues. Following pro-fibrotic stimulation using TGFß, cardiac microtissues recapitulated hallmarks of cardiac fibrosis, including myofibroblast activation and collagen deposition. A study of Ca2+ dynamics in fibrotic microtissues using optical mapping revealed prolonged Ca2+ decay, reflecting cardiomyocyte dysfunction, which is linked to the severity of fibrosis. This phenotype could be reversed by TGFß receptor inhibition or by using the BET bromodomain inhibitor, JQ1. Discussion: In conclusion, we present a novel methodology for the generation of chamber-specific cardiac microtissues that is highly scalable and allows for the multi-parametric assessment of cardiac remodelling and pharmacological screening.

Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies.

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow-stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFß stimulation and engraftment with myelofibrosis but not healthy donor-derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow-like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics. See related commentary by Derecka and Crispino, p. 263. This article is highlighted in the In This Issue feature, p. 247.

Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function.

An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.

Platelet glycoprotein VI and C-type lectin-like receptor 2 deficiency accelerates wound healing by impairing vascular integrity in mice.

Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.

The podoplanin-CLEC-2 axis inhibits inflammation in sepsis.

Platelets play a critical role in vascular inflammation through the podoplanin and collagen/fibrin receptors, C-type-lectin-like-2 (CLEC-2) and glycoprotein VI (GPVI), respectively. Both receptors regulate endothelial permeability and prevent peri-vascular bleeding in inflammation. Here we show that platelet-specific deletion of CLEC-2 but not GPVI leads to enhanced systemic inflammation and accelerated organ injury in two mouse models of sepsis-intra-peritoneal lipopolysaccharide and cecal ligation and puncture. CLEC-2 deficiency is associated with reduced numbers of podoplanin-expressing macrophages despite increased cytokine and chemokine levels in the infected peritoneum. Pharmacological inhibition of the interaction between CLEC-2 and podoplanin regulates immune cell infiltration and the inflammatory reaction during sepsis, suggesting that activation of podoplanin underlies the anti-inflammatory action of platelet CLEC-2. We suggest podoplanin-CLEC-2 as a novel anti-inflammatory axis regulating immune cell recruitment and activation in sepsis.

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse.

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac.

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

In situ reconstruction of humoral immune response against sperm: comparison of SCID and NOD/SCID mouse models.

PROBLEM: Comparison of two types of immunocompromised mouse strains (SCID and NOD/SCID) for production of human antisperm antibodies (AsA). METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were grafted to mouse peritoneal cavity and sensitized with natively glycosylated and N-deglycosylated sperm extracts. RESULTS AND CONCLUSION: NOD/SCID mice inoculated with hu-PBLs exhibited higher AsA titres with a tendency for greater sperm agglutination than human AsA raised in SCID mouse model. A comparison between 'native' and deglycosylated sperm extracts revealed higher agglutination titres by sera induced with the latter ones. Inhibitory effect of human polyclonal AsA in sperm penetration assay, however, produced opposite results to that for agglutination. Western immunoblotting was used to evaluate reactive sperm antigens prior to and after in situ sensitization showing multiple bands different from positive reactions brought by original sera of in vivo primed AsA-positive males. It seems that in situ generated AsA recognized sperm entities different from those revealed by blood donor's sera samples.

Specific Fab fragments recovered by phage display technique recognizing human spermatozoa.

Human hybridoma cell lines are often unstable and loose ability for antibody production. Sometimes, they show low and varying levels of heavy and light chains synthesis. Therefore it is reasonable to preserve generated specificities of light and heavy chains by cloning them to phagemid vector and creating phage display library. The aim of this study was to construct phage display library of Fab fragments recognizing sperm surface antigens. The source of mRNA constituted seven hybridoma cell lines producing antisperm antibodies which was proved by ELISA, and agglutination test as well as by inhibition of sperm to penetrate hamster oocytes. Fragments of cDNA encoding kappa/lambda and gamma chains were cloned into pComb3HSS phagemid vector and amplified in XL-1Blue. The library was panned against whole unfixed sperm cells. Three positive clones selected after fourth round of panning showed heavy chain belonging to VH4 family, two of them (G28, K61) possessed lambda chain from VL2 family and one (H43) kappa chain from VK1 family. As these Fabs revealed similarities to antibodies against some proteins involved in sperm motility and cell fusion it can be suggested that these Fabs may be a cause of infertility. Finally, we proved that it is feasible to preserve specificities produced by human hybridomas using phage display technique and we recovered some Fabs which may be of diagnostic and research value, and may also have some value for contraceptive vaccine.

Differential roles of the PKC novel isoforms, PKCdelta and PKCepsilon, in mouse and human platelets.

BACKGROUND: Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we focus on the role of two novel PKC isoforms, PKCdelta and PKCepsilon, in both mouse and human platelets. PKCdelta is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCdelta undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCepsilon, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCepsilon in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRgamma-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. CONCLUSIONS: These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms delta and epsilon in human and mouse platelets and a selective role for PKCepsilon in signalling through GPVI.

G6b-B inhibits constitutive and agonist-induced signaling by glycoprotein VI and CLEC-2.

Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR gamma-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cgamma (PLCgamma). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5'-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR gamma-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation.

Percutaneous trans-coronary-venous transplantation of autologous skeletal myoblasts in the treatment of post-infarction myocardial contractility impairment: the POZNAN trial.

AIMS: Several experimental studies and the initial clinical experience have shown that autologous skeletal myoblast transplantation into the area of post-infarction left ventricular injury results in an increase in segmental contractile performance. This phase I clinical trial was designed to assess the feasibility and safety of autologous skeletal myoblast transplantation performed via a percutaneous trans-coronary-venous approach in patients with post-infarction left ventricular dysfunction. METHODS AND RESULTS: Ten patients with heart failure and presence of an akinetic or a dyskinetic post-infarction injury with no viable myocardium were included in the study. Skeletal myoblasts were obtained from a biopsy specimen and grown in cell culture. Patients were treated with prophylactic amiodarone infusion before and during the procedure, except one patient. Skeletal myoblast transplantations were performed uneventfully in nine patients using the TransAccess catheter system under fluoroscopic and intravascular ultrasound guidance. In one patient, the procedure was not performed because of the inability of appropriate coronary sinus guiding wire positioning across venous valve. In five patients, the anterior interventricular vein and in four patients, the middle cardiac vein were used to access the myocardium. Two to four intramyocardial injections 1.5-4.5 cm deep were performed in each patient, delivering up to 100 million cells in 0.4-2.5 mL of saline. During 6 months follow-up, New York Heart Association class improved in all patients and ejection fraction increased 3-8% in six out of nine cases. CONCLUSION: These data suggest the feasibility and procedural safety of myoblast transplantation performed via the trans-coronary-venous approach using the TransAccess catheter system.

Percutaneous autologous myoblast transplantation in the treatment of post-infarction myocardial contractility impairment--report on two cases.

Numerous animal experimental studies as well as the initial human experience have shown that autologous skeletal myoblast transplantation into area of post-infarction left ventricular injury results in an increase in segmental contractile performance related to contraction of cells differentiated from transplanted myoblasts. We have previously introduced skeletal myoblast transplantation performed at the time of coronary artery bypass grafting. Currently, we report the first two cases in Poland of percutaneous autologous myoblast transplantation in the treatment of post-infarction heart failure. The procedures were performed using a catheter system enabling intra-myocardial injections from the lumen of cardiac veins under intravascular ultrasound guidance. Lack of major procedural complications and expected benefits from myocardial regeneration in patients with post-infarction heart failure justify initiation of phase one clinical trial to evaluate this method.

Autologous bone marrow stem cell transplantation in acute myocardial infarction-report of two cases.

The results of numerous experimental and clinical studies evaluating transplantation of bone marrow-derived pluripotential stem cells into the area of postinfarction myocardial injury, including direct myocyte precursors, are very encouraging. We have previously reported our clinical experience with transplantation of autologous skeletal myoblasts in the treatment of postinfarction myocardial injury. Currently, we report on two cases of intracoronary autologous bone marrow - derived CD34+ stem cells transplantation during acute phase of myocardial infarction. Lack of major procedural complication and expected benefits resulting from myocardial regeneration justify the initiation of a clinical study evaluating the use of this method in the treatment of patients with myocardial infarction. Our current report is only a method description and the two first cases presentation, indicating its feasibility - evaluation of the efficacy requires future investigations.