Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow

HSC homing, quiescence, and self-renewal depend on the bone marrow HSC niche. A large proportion of solid tumor metastases are bone metastases, known to usurp HSC homing pathways to establish footholds in the bone marrow. However, it is not clear whether tumors target the HSC niche during metastasis. Here we have shown in a mouse model of metastasis that human prostate cancer (PCa) cells directly compete with HSCs for occupancy of the mouse HSC niche. Importantly, increasing the niche size promoted metastasis, whereas decreasing the niche size compromised dissemination. Furthermore, disseminated PCa cells could be mobilized out of the niche and back into the circulation using HSC mobilization protocols. Finally, once in the niche, tumor cells reduced HSC numbers by driving their terminal differentiation. These data provide what we believe to be the first evidence that the HSC niche serves as a direct target for PCa during dissemination and plays a central role in bone metastases. Our work may lead to better understanding of the molecular events involved in bone metastases and new therapeutic avenues for an incurable disease.     

Plasminogen activator inhibitor type I contributes to protective immunity during experimental Gram‐negative sepsis (melioidosis)

Summary. Background: Melioidosis is a frequent cause of sepsis in Southeast Asia caused by the Gram‐negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of plasminogen activator inhibitor type 1 (PAI‐1), an important regulator of inflammation and fibrinolysis. Objectives: In this study, we aimed to investigate the role of PAI‐1 during melioidosis. Methods: Wild‐type (WT) and PAI‐1‐deficient (PAI‐1–/1^?/?) mice were intranasally infected with B. pseudomallei. Mice were killed after 24, 48 or 72 h. Lungs, liver and blood were harvested for measurement of bacterial loads, cytokines, clinical chemistry, histopathology, and coagulation parameters. Additionally, survival studies were performed. Results: PAI‐1^?/? mice demonstrated enhanced susceptibility to B. pseudomallei infection, as shown by a strongly increased mortality rate (100% vs. 58% among WT mice, P < 0.001), associated with enhanced bacterial loads in lungs, liver, and blood. Additionally, PAI‐1^?/? mice showed elevated levels of proinflammatory cytokines in lungs and plasma, accompanied by enhanced local and systemic coagulation activation (thrombin–antithrombin complexes and D‐dimer), increased hepatocellular injury (plasma aspartate aminotransferase and alanine aminotransferase), and renal failure (plasma creatinine and urea). Conclusions: PAI‐1 has a protective role during severe Gram‐negative sepsis caused by B. pseudomallei by limiting bacterial growth, inflammation, and coagulation, and probably, as a consequence thereof, distant organ injury.     

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Essentials

• Obesity is a potential risk factor for development of thrombotic thrombocytopenic purpura (TTP).
• Obese ADAMTS‐13‐deficient mice were triggered with von Willebrand factor (VWF).
• Depletion of hepatic and splenic macrophages protects against thrombocytopenia in this model.
• VWF enhances phagocytosis of platelets by macrophages, dose‐dependently.

Summary

Background

Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS‐13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets.

Objectives

To evaluate the role of obesity and ADAMTS‐13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia.

Methods

Lean or obese ADAMTS‐13‐deficient (Adamts‐13^?/?) and wild‐type (WT) mice were injected with 250 U kg^?1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts‐13^?/? mice by injection of clodronate‐liposomes, 48 h before injection of rVWF.

Results

Obese Adamts‐13^?/? mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts‐13^?/? mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts‐13^?/? mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts‐13^?/? mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose‐dependent increase of platelet phagocytosis.

Conclusions

Obese Adamts‐13^?/? mice are more susceptible to the induction of TTP‐related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

     

Factor XI promotes hemostasis in factor IX‐deficient mice

Essentials

• Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model.
• FIX‐deficient mice displayed a hemostatic defect and FXI‐deficient mice were similar to wild type mice.
• Infusion of FXI or over‐expression of FXI in FIX‐deficient mice improved hemostasis.
• FXI may affect the phenotype of FIX‐deficiency (hemophilia B).

Summary

Background

In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI‐deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI‐deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model.

Objectives

To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B).

Methods

Wild‐type mice and mice lacking either FIX (F9^?) or FXI (F11^?/?) were tested in the SVB model. The plasma levels of FXI in F11^?/? mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection.

Results

F9^? mice showed a significant defect in the SVB model, whereas F11^?/? mice and wild‐type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9^? mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX‐binding site also improved hemostasis in F9^? mice.

Conclusions

Although we were unable to demonstrate a hemostatic defect in F11^?/? mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9^? mice through FIX‐independent pathways.

     

Targeted gene inactivation reveals a functional role of calpain‐1 in platelet spreading

Summary. Background: Calpains are implicated in a wide range of cellular functions including the maintenance of hemostasis via the regulation of cytoskeletal modifications in platelets. Objectives: Determine the functional role of calpain isoforms in platelet spreading. Methods and Results: Platelets from calpain‐1^?/? mice show enhanced spreading on collagen‐ and fibrinogen‐coated surfaces as revealed by immunofluorescence, differential interference contrast (DIC) and scanning electron microscopy. The treatment of mouse platelets with MDL, a cell permeable inhibitor of calpains 1/2, resulted in increased spreading. The PTP1B‐mediated enhanced tyrosine dephosphorylation in calpain‐1^?/? platelets did not fully account for the enhanced spreading as platelets from the double knockout mice lacking calpain‐1 and PTP1B showed only a partial rescue of the spreading phenotype. In non‐adherent platelets, proteolysis and GTPase activity of RhoA and Rac1 were indistinguishable between the wild‐type (WT) and calpain‐1^?/? platelets. In contrast, the ECM‐adherent calpain‐1^?/? platelets showed higher Rac1 activity at the beginning of spreading, whereas RhoA was more active at later time points. The ECM‐adherent calpain‐1^?/? platelets showed an elevated level of RhoA protein but not Rac1 and Cdc42. Proteolysis of recombinant RhoA, but not Rac1 and Cdc42, indicates that RhoA is a calpain‐1 substrate in vitro. Conclusions: Potentiation of the platelet spreading phenotype in calpain‐1^?/? mice suggests a novel role of calpain‐1 in hemostasis, and may explain the normal bleeding time observed in the calpain‐1^?/? mice.     

Sonic Hedgehog Expands Diaphyseal Trabecular Bone Altering Bone Marrow Niche and Lymphocyte Compartment

Bone marrow contains distinct microenvironments that regulate hematopoietic stem cells (HSCs). The endosteal HSC niche includes osteoblasts, mineral, and extracellular matrix proteins that interact through various molecular signals to control HSCs. Sonic hedgehog (Shh) is a morphogen involved in the regulation of skeletal development and hematopoiesis, but the effects of Shh on bone in relation to the HSC niche are not well understood. We demonstrate that systemic overexpression of Shh in mice increases osteoblast number with the resultant formation of new trabeculae in the femoral diaphysis. Suggestive of a functional change in the hematopoietic niche, numbers of Lin⁻ Sca-1⁺ c-Kit⁺ cells with hematopoietic progenitor function expand, although cells with in vivo repopulating capacity in the wild-type environment do not increase. Instead, Shh mediates a decrease in number of bone marrow lymphocytes accompanied by a decreased expression of stromal-derived growth factor 1 (SDF-1) and a decrease in Flk2-expressing Lin⁻ Sca-1⁺ c-Kit⁺ cells, indicating a modulation of early lymphopoiesis. This is caused by a microenvironment-induced mechanism as Shh treatment of bone marrow recipients, but not donors, results in a dramatic depletion of lymphocytes. Together, these data suggest that Shh mediates alterations in the bone marrow hematopoietic niche affecting the early lymphoid differentiation.     

Factor XII activation is essential to sustain the procoagulant effects of particulate matter

See also Mutch NJ. Emerging roles for factor XII in vivo. This issue, pp 1355–8. Summary. Background: Particulate matter (PM) is a key component of ambient air pollution and has been associated with an increased risk of thrombotic events and mortality. The underlying mechanisms remain unclear. Objectives: To study the mechanisms of PM‐driven procoagulant activity in human plasma and to investigate mainly, the coagulation driven by ultrafine particles (UFPs; < 0.1 μm) in genetically modified mice. Methods: Thrombin generation in response to PM of different sizes was assessed in normal human platelet‐poor plasma, as well as in plasmas deficient in the intrinsic pathway proteases factors XII (FXII) or XI (FXI). In addition, UFPs were intratracheally instilled in wild‐type (WT) and FXII‐deficient (FXII^?/?) mice and plasma thrombin generation was analyzed in plasma from treated mice at 4 and 20 h post‐exposure. Results: In normal human plasma, thrombin generation was enhanced in the presence of PM, whereas PM‐driven thrombin formation was completely abolished in FXII‐ and FXI‐deficient plasma. UFPs induced a transient increase in tissue factor (TF)‐driven thrombin formation at 4 h post‐instillation in WT mice compared with saline instillation. Intratracheal instillation of UFPs resulted in a procoagulant response in WT mice plasma at 20 h, whereas it was entirely suppressed in FXII^?/? mice. Conclusions: Overall, the data suggest that PM promotes its early procoagulant actions mostly through the TF‐driven extrinsic pathway of coagulation, whereas PM‐driven long lasting thrombogenic effects are predominantly mediated via formation of activated FXII. Hence, FXII‐driven thrombin formation may be relevant to an enhanced thrombotic susceptibility upon chronic exposure to PM in humans.     

Pulmonary hypertension is ameliorated in mice deficient in thrombin‐activatable fibrinolysis inhibitor

Summary. Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin‐activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. Methods and results: PH was induced in C57/Bl6 wild‐type (WT) or TAFI‐deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg^?1 monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle‐to‐left ventricle‐plus‐septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI‐deficient MCT‐treated and wild‐type MCT‐treated mice suffered similar weight loss. TAFI‐deficient MCT‐treated mice had reduced levels of total protein and tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), transforming growth factor‐β (TGF‐β) and monocyte chemoattractant protein‐1 (MCP‐1) in bronchial alveolar lavage compared with wild‐type MCT‐treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT‐treated WT mice compared with MCT‐treated TAFI‐deficient mice. Conclusions: TAFI‐deficient MCT‐treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT‐treated WT animals, possibly as a result of reduced coagulation activation.     

COX‐1+/−COX‐2−/− genotype in mice is associated with shortened time to carotid artery occlusion through increased PAI‐1

Summary. Background: We found a high incidence of thrombotic deaths in COX‐1^+/?COX‐2^?/? mice and sought to define the mechanism of these events. The cyclooxygenase products thromboxane A₂ and prostacyclin are important in the regulation of coagulation but their role in fibrinolysis is largely unexplored. PAI‐1 blocks fibrinolysis by inhibiting plasminogen activator. Aim: Our objective was to explain the mechanism of increased thrombosis associated with the COX‐1^+/?COX‐2^?/? genotype. Methods: Carotid artery occlusion times were measured after photochemical injury. PAI‐1 levels were measured in the plasma by ELISA. PAI‐1 levels in the aorta were measured by RT‐PCR and Western blotting. Urinary metabolites of Thromboxane A₂ and prostacyclin were measured by ELISA. Results: The COX‐1^+/?COX‐2^?/? genotype is associated with a decreased time to occlusion in the carotid artery thrombosis model (30 ± 5 minutes vs 60 ± minutes in wild type, p<.001). The COX‐1^?/?COX‐2^+/+, COX‐1^+/?COX‐2^+/? and COX‐1^+/? COX‐2^+/+ all had occlusion times similar to wild type. COX‐1^+/+ COX‐2^?/? had a prolonged occlusion time. COX‐1^+/? COX‐2^?/? had increased PAI‐1 levels in the plasma and aorta and with a prolonged euglobulin lysis time (37.4 ± 10.2 hours vs 15.6 ± 9.8 hours in wild type, p<.004). The decreased time to occlusion in the COX‐1^+/?COX2^?/? mice was normalized by an inhibitory antibody to PAI‐1 whereas the antibody had no effect on the time to occlusion in wild type mice. Conclusion: The COX‐1^+/?COX‐2^?/? genotype is associated with a shortened time to occlusion in the carotid thrombosis model and the shortened time to occlusion is mediated through increased PAI‐1 levels resulting in decreased fibrinolysis.     

Novel insights into the development of atherosclerosis in hemophilia A mouse models

Summary. Background: Cardiovascular diseases in aging people with hemophilia (PWH) represent a growing concern. The underlying hypocoagulability probably provides a protective effect against acute thrombus formation, but the limited data available show no preventive effect against the development of atherogenesis in PWH. Atherosclerosis‐prone mice are attractive tools for the study of atherosclerosis development, and may provide insights into disease progression in PWH. Methods: Severe hemophilia A (factor VIII‐deficient [FVIII^o]) mice were crossed with mice lacking apolipoprotein E (ApoE^?/?) or mice lacking the LDL receptor (LDLR^?/?), and then compared to hemostatically normal littermate controls. After mice had received atherogenic diets for 8, 22 or 37 weeks, atherosclerotic lesion size and phenotypic characterization were analyzed in the aortic sinus and whole aortas. Results: ApoE^?/?/FVIII^o mice showed a time‐dependent protective effect against the development of atherosclerosis, beginning after 22 diet‐weeks and persisting to 37 diet‐weeks in both the aorta sinus and whole aorta as compared with ApoE^?/? mice. Notably, the FVIII deficiency did not influence the progression of atherosclerosis in the FVIII^o/LDLR^?/? model as compared with controls at early or late time points. Conclusions: Hypocoagulability ameliorates vascular disease in the ApoE‐deficient model in a lipid‐independent manner. Interestingly, FVIII deficiency did not affect the development of atherosclerosis in LDLR^?/? mice. In contrast to the ApoE model, the LDLR model resembles the lipid profile that is commonly observed in humans with atherosclerosis. These findings, to a certain extent, support the notion of atherosclerosis development in the complete absence of FVIII.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

A platelet tetraspanin superfamily member,CD151, is required for regulation of thrombus growth and stability in vivo

Summary. Background: This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. Methods and Results: Using intravital microscopy and ferric chloride (FeCl₃) injury of mesenteric arterioles, we found that thrombi formed in CD151^+/? and CD151^?/? mice were smaller and less stable, than those formed in CD151^+/+ mice, with a tendency for embolization. Similarly, in Folt’s FeCl₃?induced carotid injury model, both CD151^+/? and CD151^?/? mice showed more prolonged times to 95% vessel occlusion than CD151^+/+ mice. In addition, laser‐induced injury of cremaster muscle arterioles showed that thrombi formed in CD151^+/? and CD151^?/? mice were smaller and less stable than those formed in CD151^+/+ mice. Following platelet depletion/reconstitution with ex vivo‐labeled donor platelets, platelet‐depleted CD151^+/+ mice that received reconstitution with CD151^?/? platelets had smaller thrombi that were unstable and embolized. In contrast, platelet‐depleted CD151^?/? mice that received reconstitution with CD151^+/+ platelets had normal thrombi that were stable. Conclusions: These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.     

Reconstruction of hepatic stellate cell‐incorporated liver capillary structures in small hepatocyte tri‐culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC‐incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri‐culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell–cell interactions, which are essential to form HSC–EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d , l ‐lactide‐co‐glycolide) (PLGA) microporous membranes in SH–HSC–EC tri‐culture to reconstruct the HSC‐incorporated liver capillary structures in vitro. First, the formation of EC capillary‐like structures was induced on Matrigel‐coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary‐like structures. This process was mediated in part by platelet‐derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary‐like structures with their long cytoplasmic processes. In the HSC‐incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of hematopoietic stem cell trafficking and mobilization by the endocannabinoid system

The cannabinoid receptors CB(1) and CB(2) are seven-transmembrane Gαi protein-coupled receptors and are expressed in certain mature hematopoietic cells. We recently showed that these receptors are expressed in murine and human hematopoietic stem cells (HSCs) and that CB(2) agonists induced chemotaxis, enhanced colony formation of marrow cells, as well as caused in vivo mobilization of murine HSCs with short- and long-term repopulating abilities. Based on these observations, we have further explored the role of CB(2) and its agonist AM1241 on hematopoietic recovery following sublethal irradiation in mice. Cannabinoid receptor 2 knockout mice (Cnr2(-/-) deficient mice) exhibited impaired recovery following sublethal irradiation as compared with irradiated wild-type (WT) mice, as determined by low colony-forming units and low peripheral blood counts. WT mice treated with CB(2) agonist AM1241 following sublethal irradiation demonstrated accelerated marrow recovery and increased total marrow cells (approximately twofold) and total lineage- c-kit(+) cells (approximately sevenfold) as well as enhanced HSC survival as compared with vehicle control-treated mice. When the CB(2) agonist AM1241 was administered to WT mice 12 days before their sublethal irradiation, analysis of hematopoiesis in these mice showed decreased apoptosis of HSCs, enhanced survival of HSCs, as well as increase in total marrow cells and c-kit+ cells in the marrow. Thus, CB(2) agonist AM1241 promoted recovery after sublethal irradiation by inhibiting apoptosis of HSCs and promoting survival, as well as enhancing the number of HSCs entering the cell cycle.     

CCR2 mediates hematopoietic stem and progenitor cell trafficking to sites of inflammation in mice

HSCs are BM-derived, self-renewing multipotent cells that develop into circulating blood cells. They have been implicated in the repair of inflamed parenchymal tissue, but the signals that regulate their trafficking to sites of inflammation are unknown. As monocytes are recruited to sites of inflammation via chemoattractants that activate CCR2 on their surface, we investigated whether HSCs are also recruited to sites of inflammation through CCR2. Initial analysis indicated that in mice, CCR2 was expressed on subsets of HSCs and hematopoietic progenitor cells (HPCs) and that freshly isolated primitive hematopoietic cells (Lin^–c-Kit⁺ cells) responded to CCR2 ligands in vitro. In vivo analysis indicated that after instillation of thioglycollate to cause aseptic inflammation and after administration of acetaminophen to induce liver damage, endogenous HSCs/HPCs were actively recruited to the peritoneum and liver, respectively, in WT but not Ccr2^–/– mice. HSCs/HPCs recovered from the peritoneum successfully engrafted into the BM of irradiated primary and secondary recipients, confirming their self renewal and multipotency. Importantly, administration of exogenous WT, but not Ccr2^–/–, HSCs/HPCs accelerated resolution of acetaminophen-induced liver damage and triggered the expression of genes characteristic of the macrophage M2 or repair phenotype. These findings reveal what we believe to be a novel role for CCR2 in the homing of HSCs/HPCs to sites of inflammation and suggest new functions for chemokines in promoting tissue repair and regeneration.     

Interleukin‐10 negatively modulates extracellular signal‐regulated kinases 1 and 2 in aorta from hypertensive mouse induced by angiotensin II infusion

The activation of extracellular signal‐regulated kinase 1 and 2 (ERK 1/2) pathway promotes increased vascular contractility in angiotensin II (Ang II)‐induced hypertensive mice. Interleukin‐10 (IL‐10) is an immune‐regulatory cytokine with the ability to prevent vascular hypercontractility during hypertension. We hypothesized that IL‐10 would downregulate vascular ERK 1/2 activation during Ang II‐induced hypertension. Wild‐type (WT) or IL‐10 knockout (IL‐10^?/?) mice received Ang II infusion (90 ηg.min) or vehicle (saline), via osmotic mini‐pumps (0.25 μL/h for 14 days), whereas another WT group were infused with exogenous IL‐10 (0.5 ηg/min, 14 days) simultaneously, or not, with Ang II. Aortic rings were mounted in a myograph, and concentration‐response curves to phenylephrine were evaluated, in the presence or absence of ERK 1/2 inhibitor (PD98059, 10 μm , 40 min). Protein expression of vascular ERK 1/2 was determined by Western blot. Ang II infusion increased the maximal contractile response in both WT and IL‐10^?/? mice. Concomitant infusion of IL‐10 and Ang II prevented hypercontractility in the vasculature. Exogenous IL‐10 infusion prevented ERK 1/2 activation and hypercontractility, induced by Ang II. These findings suggest that IL‐10 negatively modulates ERK 1/2 activation and prevents hypercontractility during Ang II‐induced hypertension.     

Plasmin‐dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia

Summary. Background: The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin‐mediated cleavage of the inhibitor. Objective: To investigate the effect of plasmin generation on lung‐associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. Methods: Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue‐type plasminogen (Plg) activator (t‐PA) or Plg, at 2 h after LPS or saline injection. Results: The sharp loss of lung‐associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t‐PA^?/? and Plg^?/? mice, which are unable to generate plasmin. Conclusion: The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications.     

Ash1l controls quiescence and self-renewal potential in hematopoietic stem cells

Rapidly cycling fetal and neonatal hematopoietic stem cells (HSCs) generate a pool of quiescent adult HSCs after establishing hematopoiesis in the bone marrow. We report an essential role for the trithorax group gene absent, small, or homeotic 1-like (Ash1l) at this developmental transition. Emergence and expansion of Ash1l-deficient fetal/neonatal HSCs were preserved; however, in young adult animals, HSCs were profoundly depleted. Ash1l-deficient adult HSCs had markedly decreased quiescence and reduced cyclin-dependent kinase inhibitor 1b/c (Cdkn1b/1c) expression and failed to establish long-term trilineage bone marrow hematopoiesis after transplantation to irradiated recipients. Wild-type HSCs could efficiently engraft when transferred to unirradiated, Ash1l-deficient recipients, indicating increased availability of functional HSC niches in these mice. Ash1l deficiency also decreased expression of multiple Hox genes in hematopoietic progenitors. Ash1l cooperated functionally with mixed-lineage leukemia 1 (Mll1), as combined loss of Ash1l and Mll1, but not isolated Ash1l or Mll1 deficiency, induced overt hematopoietic failure. Our results uncover a trithorax group gene network that controls quiescence, niche occupancy, and self-renewal potential in adult HSCs.     

Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long‐term culture of HSPCs. In this study, a novel three‐dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34⁺CD38^?) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long‐term culture of primitive HSPCs in a microfluidic environment.