The roles of platelet clec-2 and podoplanin in skin wound healing

Platelet-expressed C-type lectin-like receptor-2 (CLEC-2) and glycoprotein (GP)VI play important roles in inflammation, in particular inflammatory haemostasis in the skin. The CLEC-2-ligand, podoplanin, is upregulated in the inflamed and wounded skin, but the role of the CLEC-2-podoplanin interaction and the signalling downstream of podoplanin in the repair process is unclear. I have addressed these questions by investigating skin wound healing in wild-type (WT) mice, transgenic mice that lack platelet GPVI or CLEC-2 or both receptors (double knockout; DKO), and podoplanin cytoplasmic tail-deficient (PdpnCyto) mice. Deletion of both CLEC-2 and GPVI impairs vascular integrity in the skin resulting in accelerated wound healing. The beneficial effect was due to increased plasma leakage in the tissue that promoted fibrin generation, enhanced re-epithelialisation and angiogenesis, and decreased immune cell infiltration. Accelerated wound healing also led to smaller scar formation. This healing phenotype is not due to developmental defects in DKO animals as similar results were obtained in podoplanin-blocking antibody-injected GPVI-deficient mice. Wound healing is independent of the signalling downstream of podoplanin as PdpnCyto mice had similar healing kinetics compared to WT mice. PdpnCyto mice were however capable of upregulating podoplanin during wound healing, suggesting further application of this model in inflammatory settings. Alongside wound repair, the PdpnCyto mice were characterised. I have shown that the cytoplasmic tail is dispensable for the separation of blood and lymphatic vessels. In addition, I have used a metabolomics approach to reveal an increase in M1 pro-inflammatory metabolites, i.e. glycolysis and inducible nitric oxide synthase (iNOS)-mediated arginine pathway, in bone marrow-derived podoplanin-deficient macrophages, which possibly support the anti-inflammatory activity of podoplanin in macrophages

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

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

The podoplanin-CLEC-2 axis inhibits inflammation in sepsis

Sepsis is a life-threatening condition where exaggerated inflammatory responses lead to severe tissue damage. Here, Rayes and colleagues show that the interaction between podoplanin and its receptor CLEC-2 on platelets plays a critical role in limiting inflammation during 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. </jats:p

อิทธิพลของความร้อนต่อสมบัติทางกายภาพของจีโอพอลิเมอร์คอนกรีตที่มีมวลรวมรีไซเคิลเป็นส่วนผสมInfluence of Heat on Physical Property of Geopolymer Concrete Containing Recycled Aggregates

บทความนี้ศึกษาความสามารถในการทนไฟของจีโอโพลิเมอร์คอนกรีตที่ใช้สารละลายโซเดียมไฮดรอกไซด์ต่อสารละลายโซเดียมซิลิเกตเป็นตัวกระตุ้นและใช้มวลรวมจากวัสดุรีไซเคิล การเตรียมจีโอโพลิเมอร์คอนกรีตจะใช้อัตราส่วนสารละลายต่อเถ้าลอยเท่ากับ 0.86 และอัตราส่วนสารละลายโซเดียมไฮดรอกไซด์ต่อสารละลายโซเดียมซิลิเกตเท่ากับ 1.00 ตัวอย่างจีโอโพลิเมอร์คอนกรีตที่ได้แบ่งออกเป็น 2 กลุ่มคือ ตัวอย่างที่มีการบ่มร้อนที่อุณหภูมิ 60 องศาเซลเซียส เป็นเวลา 48 ชั่วโมง และกลุ่มที่ 2 ไม่มีการบ่มร้อน ทำการทดสอบคุณสมบัติทางกลที่อายุ 28 วัน ได้แก่ค่ากำลังรับแรงอัด สัมประสิทธิ์การนำความร้อน ความต้านทานการขัดสี และความสมบูณ์ของตัวอย่าง นอกจากนี้ยังได้ทดสอบความสามารถในการทนไฟโดย ทำการเผาในเตาที่อุณหภูมิเท่ากับ 200 400 และ 600 องศาเซลเซียส เป็นเวลา 2 ชั่วโมง โดยมีอัตราการเพิ่มขึ้นของอุณหภูมิเท่ากับ 5 องศาเซลเซียสต่อนาที ผลการทดสอบพบว่าค่ากำลังรับแรงอัด สัมประสิทธิ์การนำความร้อน ความต้านทานการขัดสี และความสมบูณ์ของตัวอย่างมีค่าลดลงเมื่ออุณหภูมิในการเผาเพิ่มขึ้น และการเผาที่ 200 องศาเซลเซียส กำลังรับแรงอัดยังคงมีค่าใกล้เคียงกับตัวอย่างที่ยังไม่ผ่านกระบวนการเผา นอกจากนั้นยังพบว่าจีโอโพลิเมอร์คอนกรีตที่ผ่านการเผาที่อุณหภูมิเท่ากับ 600 องศาเซลเซียส ไม่เหมาะแก่การใช้งานที่ต้องรับกำลังและการยึดเหนี่ยวสูงThis research paper presents the heat resistance of geopolymer concrete containing recycled material by firing at maximum to 600 degree Celsius. Geopolymer concrete made by using the liquid to fly ash ratio of 0.86 and a ratio of sodium hydroxide to sodium silicate solution was 1.00. Geopolymer concrete samples were categorized into two groups: hot cured at 60 degree Celsius for 48 h and without hot curing. At the age of 28 days, the mechanical test was performed to determine the compressive strength, coefficient of thermal conductivity, abrasion resistance, and the uniformity of the samples. The sample heating was furnaced at temperature of 200, 400 and 600 degree Celsius for 2 h with a rising rate of 5 degree Celsius/minute. The results found that the compressive strength, coefficient of thermal conductivity, abrasion resistance, and the uniformity of the sample relatively decrease when firing temperature increased. And firing at 200 degree Celsius, geopolymer concrete can maintain strength by showing the equally compressive strength to unfiring. In addition, it was found that temperature over than 600 degree Celsius significantly negative affect to geopolymer concrete which further unsuitable for high strength and bonding applications

Effect of iron overload on furin expression in wild-type and β-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 β-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 β-thalassemia. Wild-type (WT) and β-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

Supplementary Material for: Effects of Iron Chelators on Pulmonary Iron Overload and Oxidative Stress in β-Thalassemic Mice

<b><i>Aim:</i></b> To evaluate the effect of iron chelators on iron-related pulmonary pathology and oxidative stress in an animal model of β-thalassemia. <b><i>Methods:</i></b> Pulmonary iron overload was induced in heterozygous β-globin knockout mice (^muβ^th-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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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