Glenzocimab: A GPVI (Glycoprotein VI)-Targeted Potential Antiplatelet Agent for the Treatment of Acute Ischemic Stroke.

It has previously been shown in several animal experiments that platelet GPVI (glycoprotein VI) contributes to thrombosis, particularly in ischemic stroke. Moreover, GPVI levels are upregulated in stroke patients. This review describes the therapeutic roles of anti-GPVI antibody in preclinical models of ischemic stroke and provides the current evidence for potential benefits of glenzocimab, a Fab fragment of humanized anti-GPVI monoclonal antibody, in stroke patients. Anti-GPVI antibody, JAQ1, significantly decreased infarct volume and improved neurological function in mice with transient middle cerebral artery occlusion, a model of ischemic stroke, with no or minor bleeding tendency. Intravenous injection of glenzocimab in nonhuman primates produced rapid inhibition of ex vivo platelet aggregation induced by collagen (a GPVI ligand). Complete platelet inhibition is observed at 30 minutes following administration without increasing the risk of bleeding. In humans, glenzocimab is well tolerated and produces dose-dependent antiplatelet activity. More importantly, glenzocimab (125-1000 mg) was safe when administered as soon as possible (<3 hours) following reperfusion with the r-tPA (recombinant tissue-type plasminogen activator) in patients with acute ischemic stroke. Although glenzocimab 1000 mg (a selected dose) did not demonstrate a significant improvement in overall clinical outcomes, it appeared to provide benefits in severe cases and in patients who required thrombectomy. This promising efficacy together with a good safety profile of glenzocimab warrant further investigation in phase III (ACTISAVE [Adaptive Efficacy and Safety Study of Glenzocimab Used as an Add-On Therapy on Top of Standard of Care in the 4.5 Hours Following an Acute Ischemic Stroke]) clinical study.

Alterations of sodium-hydrogen exchanger 1 function in response to SGLT2 inhibitors: what is the evidence?

This review summarizes and describes the current evidence addressing how sodium-glucose cotransporter 2 (SGLT2) inhibitors alter the function of sodium-hydrogen exchanger 1 (NHE-1), in association with their protective effects against adverse cardiovascular events. In the heart, SGLT2 inhibitors modulate the function of NHE-1 (either by direct inhibition or indirect attenuation of protein expression), which promotes cardiac contraction and an enhanced energy supply, in association with improved mitochondrial function, reduced inflammation/oxidative/endoplasmic reticulum stress, and attenuated fibrosis and apoptotic/autophagic cell death. The vasodilating effect of SGLT2 inhibitors has also been proposed due to NHE-1 inhibition. Moreover, platelet-expressed NHE-1 might serve as a target for SGLT2 inhibitors, since these drugs and selective NHE-1 inhibitors produce comparable activity against adenosine diphosphate-stimulated platelet activation. Overall, it is promising that the modulation of the functions of NHE-1 on the heart, blood vessels, and platelets may act as a contributing pathway for the cardiovascular benefits of SGLT2 inhibitors in diabetes and heart failure.

Lymphatic blood filling in CLEC-2-deficient mouse models.

C-type lectin-like receptor 2 (CLEC-2) is considered as a potential drug target in settings of wound healing, inflammation, and infection. A potential barrier to this is evidence that CLEC-2 and its ligand podoplanin play a critical role in preventing lymphatic vessel blood filling in mice throughout life. In this study, this aspect of CLEC-2/podoplanin function is investigated in more detail using new and established mouse models of CLEC-2 and podoplanin deficiency, and models of acute and chronic vascular remodeling. We report that CLEC-2 expression on platelets is not required to maintain a barrier between the blood and lymphatic systems in unchallenged mice, post-development. However, under certain conditions of chronic vascular remodeling, such as during tumorigenesis, deficiency in CLEC-2 can lead to lymphatic vessel blood filling. These data provide a new understanding of the function of CLEC-2 in adult mice and confirm the essential nature of CLEC-2-driven platelet activation in vascular developmental programs. This work expands our understanding of how lymphatic blood filling is prevented by CLEC-2-dependent platelet function and provides a context for the development of safe targeting strategies for CLEC-2 and podoplanin.

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.

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.

Effects of Iron Chelators on Pulmonary Iron Overload and Oxidative Stress in ß-Thalassemic Mice.

AIM: To evaluate the effect of iron chelators on iron-related pulmonary pathology and oxidative stress in an animal model of ß-thalassemia. METHODS: Pulmonary iron overload was induced in heterozygous ß-globin knockout mice (mußth-3/+, BKO). Over a period of 2 weeks, 180 mg of iron/mouse was loaded by intraperitoneal injection of iron dextran, and subsequently treated daily via intraperitoneal with either deferoxamine (DF) or deferiprone (L1) at an equimolar concentration of iron binding (0.2 and 0.6 µmol/g body weight, respectively) for 7 days. RESULTS: Iron loading resulted in iron deposition in peribronchial regions, septa and also in alveolar macrophages with a grading score of 3. This iron burden resulted in lung epithelial injuries, fibrosis and corresponded with increased lipid peroxidation and decreased tissue catalase activity. Treatment with DF or L1 resulted in a reduction of iron-laden alveolar macrophages and decreased oxidative stress and tissue damage, showing the iron mobilizing ability of both compounds. CONCLUSION: Iron chelation therapy, with DF and L1, may protect against pulmonary damage by sequestering catalytic iron and improving oxidative status. It may be beneficial in the prevention of pulmonary complications in thalassemia.

Pyroxylin-based liquid bandage forms a mechanically active protective film to facilitate skin wound healing in mice.

Wound healing requires interplay between cells and molecules. Recent evidence has demonstrated that liquid bandages promote wound healing by forming a protective barrier against contamination, attenuating inflammation, and enhancing re-epithelialization and angiogenesis. In this study, we evaluated the wound healing activity of pyroxylin-based liquid bandage (LiQuiPlast®) in eight-week-old C57BL/6 male mice by generating a single 4 mm diameter full-thickness excisional skin wound on the dorsum. In the LiQuiPlast® group, the liquid bandage was applied on day 0 and was replaced every four days. Wound size was monitored every day for two weeks. The results showed that LiQuiPlast® was mechanically active (induced wound contraction), which promoted a significant wound size reduction (27 %-39 %, compared to the control group) on days 1-4 postinjury. In addition, a significant reduction in wound size was observed again in the LiQuiPlast® group (25 %-29 %, compared to the controls) on days 8-9 postinjury. LiQuiPlast®-treated wounds showed no scab. Immunohistochemistry analyses displayed a reduction in neutrophils and tumor necrosis factor-α levels in LiQuiPlast®-treated wounds, compared to the control group on day 4 postinjury (the inflammatory phase). In addition, LiQuiPlast®-treated mice had enhanced keratinocyte proliferation than control mice during this time. On day 13 postinjury, LiQuiPlast® significantly reduced hypertrophic scarring and enhanced expression and reorganization of collagen fiber compared to control mice. In conclusion, we show that LiQuiPlast® acts as a mechanically active protective film, which promotes moist wound healing by promoting wound contraction, no scab formation, attenuated inflammation, enhanced keratinocyte proliferation, and decreased scarring.

Functional Roles of Furin in Cardio-Cerebrovascular Diseases.

Furin plays a major role in post-translational modification of several biomolecules, including endogenous hormones, growth factors, and cytokines. Recent reports have demonstrated the association of furin and cardio-cerebrovascular diseases (CVDs) in humans. This review describes the possible pathogenic contribution of furin and its substrates in CVDs. Early-stage hypertension and diabetes mellitus show a negative correlation with furin. A reduction in furin might promote hypertension by decreasing maturation of B-type natriuretic peptide (BNP) or by decreasing shedding of membrane (pro)renin receptor (PRR), which facilitates activation of the renin-angiotensin-aldosterone system (RAAS). In diabetes, furin downregulation potentially leads to insulin resistance by reducing maturation of the insulin receptor. In contrast, the progression of other CVDs is associated with an increase in furin, including dyslipidemia, atherosclerosis, ischemic stroke, myocardial infarction (MI), and heart failure. Upregulation of furin might promote maturation of membrane type 1-matrix metalloproteinase (MT1-MMP), which cleaves low-density lipoprotein receptor (LDLR), contributing to dyslipidemia. In atherosclerosis, elevated levels of furin possibly enhance maturation of several substrates related to inflammation, cell proliferation, and extracellular matrix (ECM) deposition and degradation. Neuronal cell death following ischemic stroke has also been shown to involve furin substrates (e.g., MT1-MMP, hepcidin, and hemojuvelin). Moreover, furin and its substrates, including tumor necrosis factor-α (TNF-α), endothelin-1 (ET-1), and transforming growth factor-ß1 (TGF-ß1), are capable of mediating inflammation, hypertrophy, and fibrosis in MI and heart failure. Taken together, this evidence provides functional significance of furin in CVDs and might suggest a potential novel therapeutic modality for the management of CVDs.

Benja-ummarit induces ferroptosis with cell ballooning feature through ROS and iron-dependent pathway in hepatocellular carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Benja-ummarit (BU), a traditional Thai herbal formula, has been prescribed by traditional Thai practitioners for the treatment of liver cancer. Clinical trials of BU have shown an increase in overall survival in hepatocellular carcinoma (HCC) patients, including stage 1-3 (with or without prior standard chemotherapy) and terminal stage. The clinical outcomes differ from those of other apoptosis-based conventional chemotherapies. The molecular mechanisms underlying the anti-cancer properties of BU remain unclear. AIM OF STUDY: To investigate BU-induced ferroptosis through morphological and molecular analyses of HCC cell lines and HCC rat tissues. METHODOLOGY: Cytotoxicity of BU extract in HepG2 and HuH-7 cells, with or without LX-2 in 2D and 3D cultures, was determined through MTT assay and by observing spheroid formation, respectively, as compared to sorafenib. Morphological changes and the cellular ultrastructure of the treated cells were evaluated by light microscopy and transmission electron microscopy (TEM), respectively. In addition, alterations in ferroptosis protein markers in both cell lines and rat liver tissue were determined using western blot analysis and immunohistochemical staining, respectively. To investigate the pathways mediating ferroptosis, cells were pretreated with an iron chelator to confirm the iron-dependent ferroptosis induced by the BU extract. Intracellular ROS, a mediator of ferroptosis, was measured using a scanning ion conductance microscope (SICM). SICM was also used to determine cellular stiffness. The lipid profiles of BU-treated cells were studied using LC-MS/MS. RESULTS: The BU extract induced cell death under all HCC cell culture conditions. The BU-IC50 in HepG2 and HuH-7 were 31.24 ± 4.46 µg/mL and 23.35 ± 0.27 µg/mL, respectively as determined by MTT assay. In co-culture with LX-2, BU exhibited a similar trend of cytotoxicity in both HepG2 and HuH-7 cells. Light microscopy showed cell ballooning features with intact plasma membranes, and TEM microscopy showed mitochondrial swelling and reduced mitochondrial cristae in BU-treated cells. BU promotes intracellular iron levels by increasing DMT1 and NCOA4 expression and decreasing FTH1 expression. BU also suppressed the cellular antioxidant system by lowering CD98, NRF2, and GPX4 expression, and promoting KEAP1 expression. IHC results of HCC rat liver tissues showed the absence of DMT1 and high expression of GPX4 in the tumor area. Pre-treatment with an iron chelator partially restored cell viability and shifted the mode of cell death to a more apoptosis-like morphology in the BU-treated group. The SICM showed increased intracellular ROS levels and cellular stiffness 24 h after BU treatment. In more detail of BU-mediated ferroptosis, cellular lipid profiling revealed increased expression of 3 polyunsaturated lipids, which are highly susceptible to lipid peroxidation, in BU-treated cells. DISCUSSION: Alterations in intracellular iron levels, ROS levels, and cellular lipid composition have been previously reported in cancer cells. Therefore, targeting the iron-dependent ROS pathway and polyunsaturated lipids via BU-induced ferroptosis may be more cancer-specific than apoptosis-based cancer drugs. These observations are in accordance with the clinical outcomes of BU. The ferroptosis-inducing mechanism of BU makes it an extremely promising novel drug candidate for the treatment of HCC.

Determination of the Potential Clinical Benefits of Small Molecule Factor XIa Inhibitors in Arterial Thrombosis.

Anticoagulants are the mainstay for the prevention and treatment of thrombosis. However, bleeding complications remain a primary concern. Recent advances in understanding the contribution of activated factor XI (FXIa) in arterial thrombosis with a limited impact on hemostasis have led to the development of several FXIa-targeting modalities. Injectable agents including monoclonal antibodies and antisense oligonucleotides against FXIa have been primarily studied in venous thrombosis. The orally active small molecules that specifically inhibit the active site of FXIa are currently being investigated for their antithrombotic activity in both arteries and veins. This review focuses on a discussion of the potential clinical benefits of small molecule FXIa inhibitors, mainly asundexian and milvexian, in arterial thrombosis based on their pharmacological profiles and the compelling results of phase 2 clinical studies. The preclinical and epidemiological basis for the impact of FXIa in hemostasis and arterial thrombosis is also addressed. In recent clinical study results, asundexian appears to reduce ischemic events in patients with myocardial infarction and minor-to-moderate stroke, whereas milvexian possibly provides benefits in patients with minor stroke or high-risk transient ischemic attack (TIA). In addition, asundexian and milvexian had a minor impact on hemostasis even in combination with dual-antiplatelet therapy. Other orally active FXIa inhibitors also produce antithrombotic activity in vivo with low bleeding risk. Therefore, FXIa inhibitors might represent a new class of direct-acting oral anticoagulants (DOACs) for the treatment of thrombosis, although the explicit clinical positions of asundexian and milvexian in patients with ischemic stroke, high-risk TIA, and coronary artery disease require confirmation from the outcomes of ongoing phase 3 trials.

Anti-ageing effects of FDA-approved medicines: a focused review.

Ageing is the process generated by senescent cells, free radicals, inflammation and other relevant factors. Ageing contributes to age-related diseases that affect the quality of life. People are interested in anti-ageing intervention and many scientists attempt to search for anti-ageing medicines. This review focused on describing in vivo anti-ageing activity of US-FDA-approved drugs and found that alogliptin, canagliflozin and metformin might produce anti-ageing activity via AMPK activation. Rapamycin and canagliflozin are capable to inhibit mTOR to promote lifespan. Atracurium, carnitine and statins act as DAF-16 activators, which potentially contribute to anti-ageing activity. Hydralazine, lisinopril, rosiglitazone and zidovudine may help stabilize genomic integrity to prolong life expectancy. Other indirect mechanisms, including insulin-lowering effect by acarbose and calcium channel blocking activity by verapamil may also promote longevity. Interestingly, some drugs (i.e., canagliflozin, metformin, rapamycin and acarbose) are likely to demonstrate a lifespan-promoting effect predominantly in male animals. These pre-clinical data might provide mechanistic and phenotypic perspectives to better understand the targets of anti-ageing interventions.

Dasatinib Ointment Promotes Healing of Murine Excisional Skin Wound.

Dasatinib, a tyrosine kinase inhibitor, has been shown to produce anti-inflammatory activity and impair vascular integrity in vivo, including during skin wound healing, potentially promoting the repair process. Given that dasatinib is a lipophilic small molecule capable of penetrating skin, topical dasatinib might provide benefits in wound healing. In the present study, we investigated the impact of dasatinib ointments in skin wound healing in mice. A full thickness excisional skin wound (4 mm diameter) was generated on the shaved dorsum of eight-week-old C57BL/6 mice. Dasatinib ointment (0.1 or 0.2% w/w) or ointment base was applied twice daily (every 12 h) for 10 days. Elizabethan collars were used to prevent animal licking. The wound size was monitored daily for 14 days. The results showed that dasatinib ointments, particularly 0.1% dasatinib, promoted a 16-23% reduction in wound size (p < 0.05) during day 2 to day 6 postinjury compared to controls. Immunohistochemistry analyses demonstrated a reduction in wound neutrophils (38% reduction, p = 0.04), macrophages (47% reduction, p = 0.005), and tumor necrosis factor-α levels (73% reduction, p < 0.01), together with an induction of vascular leakage-mediated fibrin(ogen) accumulation (2.5-fold increase, p < 0.01) in the wound during day 3 postinjury (an early phase of repair) in 0.1% dasatinib-treated mice relative to control mice. The anti-inflammatory and vascular hyperpermeability activities of dasatinib were associated with an enhanced healing process, including increased keratinocyte proliferation (1.8-fold increase in Ki67+ cells, p < 0.05) and augmented angiogenesis (1.7-fold increase in CD31+ area, p < 0.05), compared to the ointment base-treated group. Following treatment with 0.2% dasatinib ointment, minor wound bleeding and scab reformation were observed during the late phase, which contributed to delayed healing. In conclusion, our data suggest that dasatinib ointment, mainly at 0.1%, promotes the repair process by reducing inflammation and producing a local and temporal vascular leakage, leading to an increase in fibrin(ogen) deposition, re-epithelialization, and angiogenesis. Therefore, topical dasatinib might be a potential novel candidate to facilitate skin wound healing.

Exendin-4 protects against high glucose-induced mitochondrial dysfunction and oxidative stress in SH-SY5Y neuroblastoma cells through GLP-1 receptor/Epac/Akt signaling.

Mitochondrial dysfunction under diabetic condition leads to the development and progression of neurodegenerative complications. Recently, the beneficial effects of glucagon-like peptide-1 (GLP-1) receptor agonists on diabetic neuropathies have been widely recognized. However, molecular mechanisms underlying the neuroprotective effects of GLP-1 receptor agonists against high glucose (HG)-induced neuronal damages is not completely elucidated. Here, we investigated the underlying mechanisms of GLP-1 receptor agonist treatment against oxidative stress, mitochondrial dysfunction, and neuronal damages under HG conditions mimicking a diabetic hyperglycemic state in SH-SY5Y neuroblastoma cells. We revealed that treatment with exendin-4, a GLP-1 receptor agonist, not only increased the expression of survival markers, phospho-Akt/Akt and Bcl-2, but also decreased the expression of pro-apoptotic marker, Bax, and reduced the levels of reactive oxygen species (ROS) defense markers (catalase, SOD-2, and HO-1) under HG conditions. The expressions of mitochondrial function associated genes, MCU and UCP3, and mitochondrial fission genes, DRP1 and FIS1, were decreased by exendin-4 compared to non-treated levels, while the protein expression levels of mitochondrial homeostasis regulators, Parkin and PINK1, were enhanced. In addition, blockade of Epac and Akt activities was able to antagonize these neuroprotective effects of exendin-4. Collectively, we demonstrated that stimulation of GLP-1 receptor propagates a neuroprotective cascade against the oxidative stress and mitochondrial dysfunction as well as augments survival through the Epac/Akt-dependent pathway. Therefore, the revealed mechanisms underlying GLP-1 receptor pathway by preserving mitochondrial homeostasis would be a therapeutic candidate to alleviate neuronal dysfunctions and delay the progression of diabetic neuropathies.

Bempedoic Acid: A New Non-statin Drug for the Treatment of Dyslipidemia.

Statins are currently the first-line drugs for managing dyslipidemia due to their substantial clinical efficacy in reducing low-density lipoprotein cholesterol (LDL-C) and the risk of atherosclerotic cardiovascular disease (ASCVD). However, many patients do not reach their LDL-C target despite taking high-dose statins and some patients are intolerant of these drugs. Therefore, an additional or alternative pharmacological intervention may be required. Bempedoic acid is a novel lipid-lowering drug recently approved for the treatment of dyslipidemia. This review describes the pharmacology of bempedoic acid and its clinical role in patients with dyslipidemia. Bempedoic acid, via its active coenzyme A (CoA) form, inhibits adenosine triphosphate (ATP)-citrate lyase, and reduces hepatic cholesterol synthesis through the mevalonate pathway. The reduction in plasma LDL-C by bempedoic acid is approximately 20%. In addition, this drug is able to lower the level of high-sensitivity C-reactive protein (hs-CRP) by 20%, which suggests anti-inflammatory activity. Bempedoic acid is well tolerated by the majority of patients. Possible common adverse drug reactions include upper respiratory tract infection, urinary tract infection and arthralgia. Serum creatinine and uric acid should be monitored since increased creatinine and hyperuricemia-associated new onset of gout and gout flares have been reported in patients taking bempedoic acid. Decreased hemoglobin levels and rare tendon ruptures have also been observed. Due to its efficacy and good safety profile, bempedoic acid might serve as a potential therapeutic alternative for the management of dyslipidemia.

Dasatinib induces loss of vascular integrity and promotes cutaneous wound repair in mice.

BACKGROUND: Inflammatory bleeding due to depletion of platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential novel mechanism to promote skin wound healing. Dasatinib inhibits a broad range of tyrosine kinases, including Src and Syk, the signaling molecules downstream of GPVI and CLEC-2. OBJECTIVES: To investigate whether dasatinib affects skin wound healing. METHODS: A single (4-mm diameter) full-thickness excisional skin wound was generated in mice. Dasatinib (5 or 10 mg/kg) or dimethyl sulfoxide (DMSO) vehicle was intraperitoneally injected daily during the first 4 days. The wound was monitored over 9 days post injury. RESULTS: Dasatinib induced loss of vascular integrity during the inflammatory phase of wound repair (day 1 to day 3 post injury), which was associated with the inhibition of platelet function stimulated by collagen and rhodocytin, the ligands for GPVI and CLEC-2, respectively. Dasatinib-treated mice, particularly at 5 mg/kg, exhibited accelerated wound closure compared to DMSO-treated controls. Transient bleeding into the wound during the inflammatory phase in dasatinib-treated mice allowed for extravasation of fibrinogen. The increased deposition of fibrinogen and fibrin in the wound on day 3 post injury was associated with the augmented progression of re-epithelialization and angiogenesis, attenuated infiltration of neutrophils and macrophages, and decreased levels of tumor necrosis factor-α (TNF-α). CONCLUSIONS: Our data show that dasatinib promotes skin wound healing, and the mechanisms include blocking GPVI- and CLEC-2-mediated platelet activation, leading to self-limited inflammatory bleeding and fibrinogen/fibrin deposition, in association with reduced inflammation, increased re-epithelialization, and enhanced 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.

Iron distribution and histopathological study of the effects of deferoxamine and deferiprone in the kidneys of iron overloaded ß-thalassemic mice.

Renal glomerular and tubular dysfunctions have been reported with high prevalence in ß-thalassemia. Iron toxicity is implicated in the kidney damage, which may be reversed by iron chelation therapy. To mimic heavy iron overload and evaluate the efficacy of iron chelators in the patients, iron dextran (180mg iron/mouse) was intraperitoneally (i.p.) injected in heterozygous ß-globin knockout mice ((mußth-3/+), BKO) and wild type mice (C57BL/6J, WT) over a period of 2 weeks, followed by daily i.p. injection of deferoxamine (DFO) or deferiprone (L1) for 1 week. In BKO mice, iron preferentially accumulated in the proximal tubule with a grading score of 0-1 and increased to grade 3 after iron loading. In contrast, iron mainly deposited in the glomerulus and interstitial space in iron overloaded WT mice. Increased levels of kidney lipid peroxidation, glomerular and medullar damage and fibrosis in iron overloaded mice were reversed by treatment with iron chelators. L1 showed higher efficacy than DFO in reduction of glomerular iron, which was supported by a significantly decreased the amount of glomerular damage. Notably, DFO and L1 demonstrated a distinct pattern of iron distribution in the proximal tubule of BKO mice. In conclusion, chelation therapy has beneficial effects in iron-overloaded kidneys. However, the defect of kidney iron metabolism in thalassemia may be a determining factor of the treatment outcome in individual patients.

Effect of iron overload on furin expression in wild-type and ß-thalassemic mice.

Furin is a proprotein convertase enzyme. In the liver, it cleaves prohepcidin to form active hepcidin-25, which regulates systemic iron homeostasis. Hepcidin deficiency is a component of several iron overload disorders, including ß-thalassemia. Several studies have identified factors that repress hepcidin gene transcription in iron overload. However, the effect of iron overload on furin, a post-translational regulator of hepcidin, has never been evaluated. The present study aimed to investigate the changes in furin and related factors in parenteral iron-overloaded mice, including those with ß-thalassemia. Wild-type (WT) and ß-thalassemia intermedia (th3/+) C57BL/6 mice were intraperitoneally injected with 9 doses of iron dextran (1 g iron/kg body weight) over 2 weeks. In the iron overload condition, our data demonstrated a significant Furin mRNA reduction in WT and th3/+ mice. In addition, the liver furin protein level in iron-overloaded WT mice was significantly reduced by 70% compared to control WT mice. However, the liver furin protein in iron-overloaded th3/+ mice did not show a significant reduction compared to control th3/+ mice. The hepcidin gene (hepcidin antimicrobial peptide gene, Hamp1) expression was increased in iron-overloaded WT and th3/+ mice. Surprisingly, the liver hepcidin protein level and total serum hepcidin were not increased in both WT and th3/+ mice with iron overload, regardless of the increase in Hamp1 mRNA. In conclusion, we demonstrate furin downregulation in conjunction with Hamp1 mRNA-unrelated pattern of hepcidin protein expression in iron-overloaded mice, particularly the WT mice, suggesting that, not only the amount of hepcidin but also the furin-mediated physiological activity may be decreased in severe iron overload condition.

Iron distribution and histopathological characterization of the liver and heart of ß-thalassemic mice with parenteral iron overload: Effects of deferoxamine and deferiprone.

The liver and heart are the major target organs for iron accumulation and iron toxicity in ß-thalassemia. To mimic the phenomenon of heavy iron overload resulting from repeated blood transfusions, a total of 180 mg of iron dextran was intraperitoneally injected into C57BL/6J mice (WT) and heterozygous ß-globin knockout mice ((mu)ß(th-3/+), BKO). The effects of deferiprone and deferoxamine in this model were investigated. The iron was distributed homogenously throughout the 4 liver lobes (left, caudate, right and median) and was present in hepatocytes, Kupffer cells and the sinusoidal space. Iron accumulation in phagocytic macrophages, recruitment of hepatic lymphocytes and nucleus membrane degeneration were observed as a result of iron overload in the WT and BKO mice. However, the expansion of hepatic extramedullary hematopoiesis was observed only in the BKO mice with iron overload. In the heart, the iron accumulated in the cardiac interstitium and myocytes, and moderate hypertrophy of the myocardial fibers and cardiac myocyte degeneration were observed. Although the total liver iron was not significantly altered by iron chelation therapy, image analysis demonstrated a difference in the efficacies of two iron chelators. The major site of chelation was the extracellular compartment, but treatment with deferiprone also resulted in intracellular iron chelation. Interestingly, iron chelators reversed the pathological changes resulting from iron overload in WT and BKO mice despite being used for only a short treatment period. We suggest that some of these effects may be secondary to the anti-inflammatory activity of the chelators.