Immunohistochemical identification of lymphatic outflow in filtering blebs after non-penetrating deep sclerectomy (NPDS)

Relevance. One of the conditions for the functional outflow pathways' formation after filtering glaucoma surgeries is the uniform moisture absorption from the filtering blebs, which is provided by the blood and, possibly, lymphatic vessels of the conjunctiva. Purpose. To conduct a structural and molecular assessment of filtering blebs in patients with open-angle glaucoma (OAG) after non-penetrating deep sclerectomy (NPDS) according to OCT data, as well as ultrastructural and immunohistochemical studies. Material and methods. A clinical study of 12 patients with primary open-angle glaucoma (POAG) after NPDS was conducted. In the long-term postoperative period (12–18 months), the level of intraocular pressure (IOP) was determined, the structure of the filtering blebs using biomicroscopy and OCT of the anterior segment of the eye was evaluated, the immunohistochemical study of the filtering blebs' tissue for the expression of podoplanin and ultrastructural analysis of the samples obtained using a laser confocal microscope LSM 710 (Zeiss) was performed. In 8 cases (group 1), these were non-functional scarred filtering blebs, IOP 24.12± 2.24 mm Hg, in 4 cases (group 2) – functional filtering blebs, IOP 15.31± 4.08 mm Hg. Results. During the immunohistochemical examination of the first group patients' conjunctival samples, no vessels with characteristics of the lymphatic system were detected in any case. In each sample of the second group patients' filtering blebs, from 5 to 7 vessels with different variants of podoplanin expression were determined. Conclusion. The aqueous outflow entering the subconjunctival space through the newly created outflow pathway during glaucoma surgery is carried out at the expense of conjunctival lymphatic structures, the condition of which to a certain extent determines the success or failure of the surgery. Key words: non-penetrating deep sclerectomy; open-angle glaucoma; lymphatic outflow; filtering blebs

CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

Chitinase 3-like-1 Contributes to Acetaminophen-induced Liver Injury by Promoting Hepatic Platelet Recruitment

Hepatic platelet accumulation contributes to acetaminophen (APAP)-induced liver injury (AILI). However, little is known about the molecular pathways involved in platelet recruitment to the liver and whether targeting such pathways could attenuate AILI. The present study unveiled a critical role of chitinase 3-like-1 (Chi3l1) in hepatic platelet recruitment during AILI. Increased Chi3l1 and platelets in the liver were observed in patients and mice overdosed with APAP. Compared to wild-type (WT) mice, Chi3l1-/- mice developed attenuated AILI with markedly reduced hepatic platelet accumulation. Mechanistic studies revealed that Chi3l1 signaled through CD44 on macrophages to induce podoplanin expression, which mediated platelet recruitment through C-type lectin-like receptor 2. Moreover, APAP treatment of CD44-/- mice resulted in much lower numbers of hepatic platelets and liver injury than WT mice, a phenotype similar to that in Chi3l1-/- mice. Recombinant Chi3l1 could restore hepatic platelet accumulation and AILI in Chi3l1-/- mice, but not in CD44-/- mice. Importantly, we generated anti-Chi3l1 monoclonal antibodies and demonstrated that they could effectively inhibit hepatic platelet accumulation and AILI. Overall, we uncovered the Chi3l1/CD44 axis as a critical pathway mediating APAP-induced hepatic platelet recruitment and tissue injury. We demonstrated the feasibility and potential of targeting Chi3l1 to treat AILI.

Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilise amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by the RhoA signalling pathway. Migrastatic drugs targeting actin polymerization and contractility to inhibit invasion and metastasis are therefore a promising treatment option. To predict amoeboid invasion and metastatic potential, there is a need for biomarkers functionally linked to contractility pathways. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts, and is overexpressed in several cancer types. Here, we show that podoplanin enhances amoeboid invasion in melanoma. Expression of podoplanin in murine melanoma models drives rounded cell morphology, increasing motility and invasion in vivo. Podoplanin expression is upregulated in a subset of dedifferentiated human melanoma, and in vitro is sufficient to suppress melanogenesis and upregulate melanoma-associated markers Mitf and Pou3f2. Together, our data indicates that podoplanin is both a potential biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

Podoplanin-mediated platelet activation promotes proliferation and invasion of colon cancer cells

Background: Recent studies have shown that podoplanin is highly expressed in many tumors, suggesting that podoplanin may be related to the invasion and metastasis of malignant tumors. A potential mechanism by which podoplanin promote tumor invasion and metastasis is podoplanin-mediated platelet aggregation and activation.Methods: The expression of podoplanin in colon cancer LOVO cell line and lung adenocarcinoma cell line A549 was detected by immunoblotting. The above cells were co-cultured with washed mouse platelets. The platelet aggregation meter was used to determine the platelet aggregation rate and observe the cell morphology. Immunofluorescence staining and immunoblotting were used to detect the expression of EMT-related factors and the phosphorylation level of Smad2/3 downstream of the TGF-β signal; and then the matrigel-coated transwell chamber invasion test was used to evaluate the invasion ability of tumor cells. Lentiviruses targeting human podoplanin shRNA were constructed to infect colon cancer LOVO cells to generate cell lines with stable podoplanin knockdown, and the above experiments were repeated. Results: The colon cancer LOVO cell line with positive podoplanin expression had platelet aggregation activity, and the platelet aggregation activity of colon cancer LOVO cell line disappeared when anti-podoplanin monoclonal antibody MS-140 was added. However, no podoplanin expression and platelet aggregation activity were detected in lung adenocarcinoma cell line A549. When colon cancer LOVO cell-platelet reactant supernatant was added to colon cancer LOVO cell culture, the morphology of colon cancer LOVO cells showed EMT characteristics. Immunoblot showed that E-cadherin was down-regulated in colon cancer LOVO cells, while Vimentin And N-cadherin expression are upregulated. However, in lung adenocarcinoma A549 cells, there were no changes in EMT morphology and EMT factors. Immunoblot and ELISA showed that colon cancer LOVO cells with positive expression of podoplanin released TGF-β after aggregating with platelets, activated the TGF-β/Smad2/3 signaling pathway, and induced EMT and increased invasiveness of colon cancer LOVO cells. Treating colon cancer LOVO cells with TGF-β antibodies and TGF-β receptor inhibitors, or using LOVO cell lines with podoplanin knockdown, and repeating the above experiments suggest that platelet aggregation activity disappears and tumor cells EMT are eliminated, and The ability to invade is reduced. Conclusions: Podoplanin-mediated platelet activation plays an important role in colon cancer invasion and metastasis; its possible mechanism is that colon cancer LOVO cells with positive podoplanin expression react with platelets, activate platelets to release TGF-β, and activate TGF-β / Smad2 / 3 Signaling pathway induces EMT in colon cancer LOVO cells and enhances tumor cell proliferation and invasiveness.