Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing

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--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 A2 (TxA2). The role of ADP and TxA2 in mediating hosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast,tyrosine phosphorylation of the GPVIFcR -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

RET mutation and increased angiogenesis in medullary thyroid carcinomas

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs. RET mutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somatic RET mutations (RETmut group) and 34 with wild-type RET (RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared with RETwt tumors, RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels in RETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis in RETmut MTCs may be more intense and complete than that found in RETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density, RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors

Reproducibility of lymphovascular space invasion (LVSI) assessment in endometrial cancer

Aims Lymphovascular space invasion (LVSI) in endometrial cancer (EC) is an important prognostic variable impacting on a patient's individual recurrence risk and adjuvant treatment recommendations. Recent work has shown that grading the extent of LVSI further improves its prognostic strength in patients with stage I endometrioid EC. Despite this, there is little information on the reproducibility of LVSI assessment in EC. Therefore, we designed a study to evaluate interobserver agreement in discriminating true LVSI from LVSI mimics (Phase I) and reproducibility of grading extent of LVSI (Phase II). Methods and results Scanned haematoxylin and eosin (H&E) slides of endometrioid EC (EEC) with a predefined possible LVSI focus were hosted on a website and assessed by a panel of six European gynaecological pathologists. In Phase I, 48 H&E slides were included for LVSI assessment and in Phase II, 42 H&E slides for LVSI grading. Each observer was instructed to apply the criteria for LVSI used in daily practice. The degree of agreement was measured using the two-way absolute agreement average-measures intraclass correlation coefficient (ICC). Reproducibility of LVSI assessment (ICC = 0.64, P < 0.001) and LVSI grading (ICC = 0.62, P < 0.001) in EEC was substantial among the observers. Conclusions Given the good reproducibility of LVSI, this study further supports the important role of LVSI in decision algorithms for adjuvant treatment

Induction of podoplanin by transforming growth factor-β in human fibrosarcoma

Podoplanin/aggrus is increased in tumors and its expression was associated with tumor malignancy. Podoplanin on cancer cells serves as a platelet-aggregating factor, which is associated with the metastatic potential. However, regulators of podoplanin remain to be determined. Transforming growth factor-β (TGF-β) regulates many physiological events, including tumorigenesis. Here, we found that TGF-β induced podoplanin in human fibrosarcoma HT1080 cells and enhanced the platelet-aggregating-ability of HT1080. TGF-β type I receptor inhibitor (SB431542) and short hairpin RNAs for Smad4 inhibited the podoplanin induction by TGF-β. These results suggest that TGF-β is a physiological regulator of podoplanin in tumor cells

Podoplanin expression in the development and progression of laryngeal squamous cell carcinomas

Background: Podoplanin expression is attracting interest as a marker for cancer diagnosis and prognosis. We therefore investigated the expression pattern and clinical significance of podoplanin during the development and progression of laryngeal carcinomas. Results: Podoplanin expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal premalignancies and 53 patients with laryngeal squamous cell carcinomas. We found podoplanin expression extending from the basal to the suprabasal layer of the epithelium in 37 (44%) of 84 dysplastic lesions, whereas normal epithelium showed negligible expression. Patients carrying podoplanin-positive lesions had a higher laryngeal cancer incidence than those with negative expression reaching borderline statistical significance (51% versus 30%, P = 0.071). Podoplanin expression in laryngeal carcinomas exhibited two distinct patterns. 20 (38%) cases showed diffuse expression in most tumour cells and 33 (62%) focal expression at the proliferating periphery of tumour nests. High podoplanin expression was inversely correlated with T classification (P = 0.033), disease stage (P = 0.006), and pathological grade (P = 0.04). There was a trend, although not significant, towards reduced disease-specific survival for patients with low podoplanin levels (P = 0.31) and diffuse expression pattern (P = 0.08). Conclusions: Podoplanin expression increases in the early stages of laryngeal tumourigenesis and it seems to be associated with a higher laryngeal cancer risk. Podoplanin expression in laryngeal squamous cell carcinomas, however, diminishes during tumour progression. Taken together, these data support a role for podoplanin expression in the initiation but not in the progression of laryngeal cancer

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the haematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other haematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that is dependent on CLEC-2 and Syk. These studies demonstrate that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behaviour in vitro

Dye diffusion during laparoscopic tubal patency tests may suggest a lymphatic contribution to dissemination in endometriosis: A prospective, observational study

Aim Women with adenomyosis are at higher risk of endometriosis recurrence after surgery. This study was to assess if the lymphatic vessel network drained from the uterus to near organs where endometriosis foci lied. Methods A prospective, observational study, Canadian Task Force Classification II-2, was conducted at Sacro Cuore Don Calabria Hospital, Negrar, Italy. 104 white women aged 18–43 years were enrolled consecutively for this study. All patients underwent laparoscopy for endometriosis and a tubal dye test was carried out. Results Evidence of dye dissemination through the uterine wall and outside the uterus was noted in 27 patients (26%) with adenomyosis as it permeated the uterine wall and a clear passage of the dye was shown in the pelvic lymphatic vessels regardless whether the tubes were unobstructed. Histological assessment of the uterine biopsies confirmed adenomyosis. Conclusion Adenomyosis is characterized by ectatic lymphatics that allow the drainage of intrauterine fluids (the dye and, perhaps, menstrual blood) at minimal intrauterine pressure from the uterine cavity though the lymphatic network to extrauterine organs. Certainly, this may not be the only explanation for endometriosis dissemination but the correlation between the routes of the dye drainage and location of endometriosis foci is highly suggestive