Lymphatic Vascular Density, the Expression of Podoplanin and Tumor Budding in Oral Squamous Cell Carcinoma.

BACKGROUND: Notwithstanding recent advances in oral squamous cell carcinoma (OSCC) management, its mortality rate is still high. It is imperative to investigate new parameters that are complementary to clinical staging for OSCC to provide better prognostic insight. The presence of isolated neoplastic cells or small clusters of up to four cells at the tumor's invasive front, called tumor budding, is a morphological marker of OSCC with prognostic value. Increased lymphatic vascular density (LVD) and a high expression of podoplanin in neoplastic cells have also been associated with worse prognosis in OSCC. To investigate these markers in OSCC, we evaluated differences in LVD and the expression of podoplanin in neoplastic cells between tumors with high-intensity tumor budding versus low-intensity or no tumor budding. In the samples of high-intensity budding, differences in those parameters between the​​ budding area and the area outside the budding were also evaluated. Furthermore, the study assessed differences in LVD and in the expression of podoplanin in neoplastic cells concerning OSCC clinicopathological characteristics. METHODS: To those ends, we subjected 150 samples of OSCC to immunohistochemistry to evaluate the intensity of tumor budding (via multi-cytokeratin immunostaining). Moreover, the 150 samples of OSCC and 15 specimens of normal oral mucosa (used as a control) were employed to assess LVD and the expression of podoplanin (in neoplastic cells of OSCC and in the lining epithelium of normal oral mucosa), both via podoplanin immunostaining. Data were processed into descriptive and analytical statistics. RESULTS: No differences were observed neither in the LVD nor in the expression of podoplanin in neoplastic cells concerning sex, age, tobacco smoking, tumor location and tumor size. The LVD was greater in OSCC and in tumors with high-intensity budding than in normal mucosa but did not differ between normal mucosa and tumors with low-intensity or no tumor budding. The data analyses also revealed that LVD was greater in tumors with high-intensity tumor budding than in tumors with low-intensity or no budding and showed no difference in LVD between the budding area and the area outside the budding. When compared to the lining epithelium of the normal mucosa, the expression of podoplanin was greater in neoplastic cells of OSCC, tumors with high-intensity budding and tumors with low-intensity or no tumor budding. The expression of podoplanin in neoplastic cells was also greater in tumors with high-intensity budding and, within those tumors, greater in the budding area than in the area outside de budding. CONCLUSION: Those findings support the hypothesis that tumor budding is a biological phenomenon associated with the progression and biological behavior of OSCC.

Avaliação imunohistoquímica dos vasos linfáticos em fígados humanos com hepatite crônica C - relação com variáveis histológicas / Immunohistochemical assessment of lymphatic vessels in human livers with chronic hepatitis c - relation to histological variables

ABSTRACT Background Viral hepatitis C is a significant public health challenge. The disease may remain clinically silent in both acute and chronic forms, and chronic infections may progress to advanced disease such as cirrhosis and hepatocellular carcinoma, requiring costly treatment, compromising the patient's quality of life and even leading to death. For this reason, it is one of the most frequent indications for liver transplantation. Although treatment with direct-acting antivirals represents remarkable progress, many patients are still infected and even those who cleared the viral infection must be followed due to their previous hepatic lesions, especially regarding the disturbances of lobular architecture and the sanguineal and lymphatic vessels. Objective To assess immunohistochemical aspects of lymphatic sprouts and mature lymphatic vascularity with histological variables of liver injury attributable to hepatitis C virus (HCV) and fatty disease. Methods The present study included 72 liver biopsies of cases with chronic hepatitis C. Morphologic changes reflecting "staging" and "activity" were analyzed. Immunohistochemical reactions were performed with monoclonal antibody D2-40 anti-podoplanin. Major histological variables were also semiquantified so as to enable the search for possible associations among histological and Immunohistochemical criteria, as well as with genotypes 1 and 3 of HCV. Results Histological findings showed that the different degrees of strutural changes were well represented in this casuistic. Intralobular/parenchymal necro-inflammatory activity was predominantly mild to moderate. Most cases did not show major evidences of fatty disease, which was found significantly higher in cases infected with HCV genotype 3. The amount of portal lymphatic sprouts increased along with the progression of structural changes, maximal at cirrhosis. Portal lymphatic sprouts as well as portal mature lymphatic vessels also showed an increase parallel to the increase in the degree of portal/septal inflammatory infiltrate. In the present study, no significant association was found between the proportion of portal lymphatic sprouts or portal mature lymphatic vessels and the degree of periportal/periseptal activity. No significant relations were detected between lymphatic sprouts/mature vessels and periportal or parenchymal inflammatory activity, nor with infections due to HCV genotype 1 or 3. Conclusion Visualization and semiquantitation of sprouts and mature lymphatic vessels were clearly yielded by Immunohistochemical staining with monoclonal antibody D2-40. The amount of lymphatics was increased along fibrogenic process, significantly related to progression of liver disease and maximal at cirrhosis. No significant relations were detected with necro-inflammatory activity at interface or in the parenchyma.

RESUMO Contexto A hepatite C é um relevante problema de saúde pública. A doença pode permanecer clinicamente silenciosa tanto na forma aguda como na crônica e as infecções crônicas podem progredir para doenças avançadas, tais como cirrose e carcinoma hepatocelular (CHC), requerendo tratamentos dispendiosos, comprometendo a qualidade de vida do paciente e até mesmo levando à morte. Por esta razão, é uma das indicações mais frequentes para o transplante hepático. Apesar da introdução do tratamento com antivirais de ação directa (AAD) representar um progresso notável, muitos pacientes não receberam o tratamento e continuam infectados, e mesmo aqueles que eliminaram a infecção viral devem ser seguidos devido às lesões hepáticas anteriores, especialmente no que diz respeito às alterações da arquitetura lobular e dos vasos sanguíneos e linfáticos. Objetivo Avaliar os aspectos imuno-histoquímicos dos brotos linfáticos e dos vasos linfáticos "maduros" com variáveis histológicas de lesão hepática atribuíveis ao vírus da hepatite C (VHC) e à doença gordurosa. Métodos O presente estudo incluiu 72 biópsias hepáticas em pacientes com hepatite C crônica. Foram analisadas alterações estruturais relativas a "estadiamento" e "atividade". Reações imuno-histoquímicas foram realizadas com anticorpo D2-40 anti-podoplanina. As principais variáveis histológicas também foram semiquantificadas, de modo a permitir a procura de possíveis associações entre os critérios histológicos e imunohistoquímicos, bem como com os genótipos 1 e 3 do VHC. Resultados Os achados histológicos mostraram que os diferentes graus de alterações estrutural estavam bem representados nesta casuística. A atividade necro-inflamatória lobular/parenquimatosa foi predominantemente leve à moderada. A maioria dos casos não apresentava grandes evidências de doença gordurosa, que foi encontrada significativamente mais elevada nos casos infectados com o genótipo 3 do VHC. A quantidade de brotos linfáticos portais aumentou com a progressão de alterações estruturais, sendo máxima na cirrose. Os brotos linfáticos portais, bem como os vasos linfáticos "maduros" portais também mostraram um aumento paralelo ao aumento do grau de infiltrado inflamatório portal/septal. No presente estudo, não foi encontrada qualquer associação significativa entre a proporção de brotos linfáticos portais ou vasos linfáticos maduros portais e o grau de atividade periportal/periseptal. Não foram detectadas relações significativas entre os brotos linfáticos/vasos maduros e a atividade inflamatória periportal ou atividade inflamatória parenquimatosa, nem com infecções devido ao genótipo 1 ou 3 do VHC. Conclusão A reação imunohistoquímica com anticorpo monoclonal D2-40 possibilitou a visualização e a semiquantitação de brotos e vasos linfáticos "maduros" nas amostras obtidas por biópsia hepática. A quantidade de linfáticos aumentou ao longo do processo fibrogênico, significativamente relacionada com a progressão da doença hepática e máxima na cirrose. Não foram detectadas relações significativas com a atividade necro-inflamatória periportal ou parenquimatosa.

Mast Cells in the Tumor Microenvironment.

Mast cells are tissue-resident, innate immune cells that play a key role in the inflammatory response and tissue homeostasis. Mast cells accumulate in the tumor stroma of different human cancer types, and increased mast cell density has been associated to either good or poor prognosis, depending on the tumor type and stage. Mast cells play a multifaceted role in the tumor microenvironment by modulating various events of tumor biology, such as cell proliferation and survival, angiogenesis, invasiveness, and metastasis. Moreover, tumor-associated mast cells have the potential to shape the tumor microenvironment by establishing crosstalk with other tumor-infiltrating cells. This chapter reviews the current understanding of the role of mast cells in the tumor microenvironment. These cells have received much less attention than other tumor-associated immune cells but are now recognized as critical components of the tumor microenvironment and could hold promise as a potential target to improve cancer immunotherapy.

The immune-checkpoint HLA-G/ILT4 is involved in the regulation of VEGF expression in clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), the most aggressive renal cancer, is characterized by early lymph node metastases and bad prognosis. Most therapies targeting advanced or metastatic ccRCC are based, as first-line treatment, on the administration of the vascular endothelial growth factor (VEGF) neutralizing antibody termed Bevacizumab. Despite proven benefits, the expected results were not obtained for the majority of patients. The possibility that an intricate interplay between angiogenesis and immune-checkpoints might exist lead us to evaluate tumor angiogenesis, by means of VEGF expression together with the immune checkpoint HLA-G/ILT4. METHODS: Tumor specimens were obtained from patients from two separate cohorts: One from "Evita Pueblo" Hospital from Berazategui, (Buenos Aires, Argentina) and the second includes patients surgically operated at the Urology Department of Saint-Louis Hospital (Paris, France) with a confirmed ccRCC diagnosis. Immunohistochemistry was performed with specific antibodies directed against HLA-G, VEGF-A, VEGF-C, D240, CD34, ILT4 and Ca-IX. In addition, gene expression levels were measured in a cell line derived from a ccRCC patient by semi-quantitative RT-PCR. RESULTS: Our results show that the highly vascularized tumors of ccRCC patients express high levels of VEGF and the immune-checkpoint HLA-G. In addition, ILT4, one of the HLA-G receptors, was detected on macrophages surrounding tumor cells, suggesting the generation of an immune-tolerant microenvironment that might favor tumorigenesis. Notably, RT-qPCR analysis provided the first evidence on the transcriptional relationship between HLA-G/ILT4 and the VEGF family. Namely, in the presence of HLA-G or ILT4, the levels of VEGF-A are diminished whereas those of VEGF-C are increased. CONCLUSIONS: In an effort to find new therapeutic molecules and fight against metastasis dissemination associated with the poor survival rates of ccRCC patients, these findings provide the rationale for co-targeting angiogenesis and the immune checkpoint HLA-G.

Matrix Metalloproteinases' Role in Tumor Microenvironment.

Cancer cells evolve in the tumor microenvironment (TME) by the acquisition of characteristics that allow them to initiate their passage through a series of events that constitute the metastatic cascade. For this purpose, tumor cells maintain a crosstalk with TME non-neoplastic cells transforming them into their allies. "Corrupted" cells such as cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and tumor-associated neutrophils (TANs) as well as neoplastic cells express and secrete matrix metalloproteinases (MMPs). Moreover, TME metabolic conditions such as hypoxia and acidification induce MMPs' synthesis in both cancer and stromal cells. MMPs' participation in TME consists in promoting events, for example, epithelial-mesenchymal transition (EMT), apoptosis resistance, angiogenesis, and lymphangiogenesis. MMPs also facilitate tumor cell migration through the basement membrane (BM) and extracellular matrix (ECM). The aim of the present chapter is to discuss MMPs' contribution to the evolution of cancer cells, their cellular origin, and their influence in the main processes that take place in the TME.

Immunohistochemical study of CD34 and podoplanin in periodontal disease.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the angiogenesis and lymphangiogenesis in gingival tissue biopsy specimens of individuals with clinically healthy gingiva, chronic gingivitis, and chronic periodontitis (n = 30 per clinical condition). MATERIAL AND METHODS: Histological sections were stained using hematoxylin and eosin as well as immunohistochemically with hematopoietic progenitor cell antigen CD34 and podoplanin (PDPN) antibodies to evaluate the microvascular count, area, and perimeter of blood and lymphatic vessels, respectively. RESULTS: The results revealed a correlation between the microvascular count of blood and lymphatic vessels (P = 0.03; however, in individuals with chronic periodontitis, fewer lymphatic vessels were present than in the clinically healthy gingival tissue (P = 0.01), which was not observed in the case of microvascular area and perimeter. Podoplanin labeling was present in the epithelium, and the intensity of labeling was positively correlated to the intensity of the inflammatory infiltrate (P = 0.03). CONCLUSION: In this study, we concluded that an increase in the number of blood and lymphatic vessels was not observed in bouth gingivitis and periodontitis samples. Podoplanin expression is highly associated with an increased inflammatory infiltration suggesting that PDPN might play an additional role in periodontal disease, other than solely as a lymphangiogenesis marker.

Immunohistochemical expression of podoplanin (D2-40), lymphangiogenesis, and neoangiogenesis in tooth germ, ameloblastomas, and ameloblastic carcinomas.

BACKGROUND: Ameloblastoma is a benign but locally aggressive odontogenic tumor, while ameloblastic carcinoma is its malignant counterpart. Angiogenesis and lymphangiogenesis in malignancies have been correlated with higher aggressiveness and poor prognosis, as well as greater expression of podoplanin by tumoral cells. METHODS: Immunohistochemical expression of podoplanin, CD34, and CD105 (endoglin) was evaluated in 53 ameloblastomas and three ameloblastic carcinomas; additionally, immunohistochemistry for podoplanin was also performed in 10 tooth germs. Microvessel density of blood and lymphatic vessels was calculated and compared between ameloblastomas and ameloblastic carcinomas. Immunoexpression of podoplanin by ameloblastic cells was evaluated in tooth germs, ameloblastomas, and ameloblastic carcinomas. RESULTS: Podoplanin was similarly expressed by odontogenic epithelial cells of tooth germs and ameloblastomas, while its expression was lower in ameloblastic carcinomas. There was no difference in microvessel density assessed by CD34 between ameloblastomas and ameloblastic carcinomas; nevertheless, the latter presented higher amounts of lymphatic and new formed blood vessels. CONCLUSIONS: Results suggest that podoplanin does not seem to be involved in invasion mechanisms of ameloblastic carcinomas, as its expression was decreased in the malignant tumoral cells. On the other hand, the increased lymphatic microvessel density and neoangiogenesis found in ameloblastic carcinomas could be related to its aggressiveness and potential for metastasis.

Análise da imunoexpressão da podoplanina e da triptase em carcinoma epidermóide de língua e sua relação com parâmetros clinicopatológicos / Immunohistochemical analysis of podoplanin and tryptase in squamous cell carcinoma of the tongue and its relationship with clinicopathological parameters

O carcinoma epidermóide de língua oral (CELO) apresenta um comportamento biológico agressivo, com elevada propensão ao desenvolvimento de metástases nodais. Nesse contexto, a linfangiogênese é considerada um fenômeno importante para a disseminação das células tumorais e pode sofrer influência de estímulos do microambiente. Os mastócitos têm sido relacionados à progressão de neoplasias malignas, no entanto o seu papel na formação de vasos linfáticos ainda não está bem estabelecido. O propósito desta pesquisa foi avaliar possíveis correlações entre a densidade linfática, a contagem de mastócitos e o perfil clinicopatológico em casos de CELO, incluindo o estadiamento clínico TNM, a gradação histológica de malignidade (Bryne, 1998) e a presença/ausência de metástases nodais. A amostra foi constituída por 50 casos de CELO, dos quais 26 apresentavam metástase nodal, e os 24 restantes eram isentos de metástases. A densidade linfática foi estabelecida como a média de vasos linfáticos imunomarcados pelo anticorpo anti-podoplanina (D2-40), identificados em cinco campos microscópicos (200x). Para a análise dos mastócitos, foram quantificadas as células imunorreativas ao anticorpo anti-triptase, em cinco campos (400x). Destaca-se que ambas as imunomarcações foram analisadas no centro tumoral e no front de invasão. A densidade linfática intratumoral (DLI) foi superior nos casos em estágios clínicos avançados (III-IV), quando comparados àqueles em estágios iniciais (I-II), assim como nos casos metastáticos em relação aos não-metastáticos (p<0,05).

Não houve diferenças estatisticamente significativas entre os casos de baixo grau e alto grau de malignidade no tocante à DLI (p>0,05). De outro modo, a densidade linfática peritumoral (DLP) e as contagens de mastócitos não demonstraram relações significativas com nenhum dos parâmetros clinicopatológicos avaliados (p>0,05). Também não foram encontradas correlações significativas entre as densidades linfáticas e as contagens de mastócitos, seja na região intratumoral (r = -0,004; p=0,977) ou na peritumoral (r = -0,154; p=0,285). Os resultados do presente estudo sugerem que os vasos linfáticos intratumorais contribuem na progressão do CELO. Por sua vez, a DLP pode não ser suficiente para justificar diferenças no comportamento biológico do CELO, o que sustenta a hipótese de envolvimento de outros mecanismos na disseminação metastática das células malignas, que complementariam os efeitos da linfangiogênese. Os mastócitos, ainda que realizem diversas funções pró- e antitumorais, parecem não influenciar diretamente o potencial de agressividade do CELO. Adicionalmente, é possível que a quantidade destas células não seja um fator determinante para a formação de vasos linfáticos. (AU)

Oral tongue squamous cell carcinoma (OTSCC) has an aggressive biological behavior, with a high propensity for the development of lymph node metastases. In this context, lymphangiogenesis is considered an important phenomenon for the spread of tumor cells and may be influenced by microenvironmental stimuli. Mast cells have been implicated in tumor progression, although their influence in the formation of lymphatic vessels is not well established. The aim of this study was to analyze, in a case series of OTSCC (n=50), possible correlations between lymphatic vessel density (LVD), mast cell count and clinicopathological features, including tumor-node-metastasis (TNM) stage, histological grade of malignancy (Bryne, 1998), and nodal metastasis. LVD was established as the mean number of lymphatic vessels immunostained by anti-podoplanin (D2-40) antibody, identified in five microscopic fields (200x). For the analysis of mast cells, tryptase-immunoreactive cells were quantified in five fields (400x). Both immunostainings were analyzed in the tumor center and invasion front. Intratumoral lymphatic density (ILD) was higher in cases in advanced clinical stages (III-IV), compared to those in initial stages (I-II), as well as in metastatic cases in respect of non-metastatic (p<0,05).

There were no statistically significant differences between low-grade and high-grade malignancy cases with respect to ILD (p>0,05). Peritumoral lymphatic density (PLD) and mast cell counts showed no significant relations with any of the clinicopathological parameters evaluated (p>0,05). Also there were no significant correlations between LVD and mast cell counts, whether in intratumoral (r = -0,004; p=0,977) or peritumoral region (r = -0,154; p=0,285). The results of the present study suggest that intratumoral lymphatic vessels may contribute in part to the progression of OTSCC, although PLD may be insufficient to justify differences in biological behavior. This supports the hypothesis of involvement of other mechanisms in metastatic spread of malignant cells, which could complement the effects of lymphangiogenesis. Although mast cells perform several pro- and antitumoral functions, they do not appear to directly influence aggressiveness of OTSCC. In addition, the quantity of these cells may not be essential for lymphatic vessel formation. (AU)

Estudo imunoistoquímico do CD34 e podoplanina na doença periodontal / Immunohistochemical study of CD34 and podoplanin in periodontal disease

A angiogênese e a linfangiogênese são alterações também decorrentes da inflamação gengival provocada por microrganismos presentes no biofilme dental, bem como pela a migração de células de defesa e secreção de mediadores inflamatórios no local da agressão. Este estudo teve por objetivo avaliar a angiogênese e linfangiogênese em 90 espécimes de biópsias de tecido gengival clinicamente saudável, com gengivite e com periodontite crônicas. Os cortes histológicos foram avaliados pela coloração de hematoxilina e eosina e pela técnica de imunoistoquímica através da imunomarcação de CD34 e podoplanina, para avaliar, respectivamente, o índice angiogênico e linfangiogênico, por meio da técnica de contagem microvascular. Os resultados mostraram que há correlação entre os índices (p=0,030), porém, mostrou que na periodontite há menos números de vasos linfáticos do que no tecido gengival clinicamente saudável (p=0,016). A podoplanina mostrou marcação no epitélio e que há relação da intensidade de marcação com a intensidade do infiltrado inflamatório, sendo mais intensa a marcação na presença de infiltrado inflamatório severo (p=0,033). Concluiu-se neste estudo que há menor número de vasos sanguíneos na periodontite em comparação com a gengiva clinicamente saudável. As sinalizações presentes no processo inflamatório, bem como o real papel da vasculatura sanguínea e linfática gengival ainda não estão totalmente elucidadas. (AU)

Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested. (AU)

Lymphangiogenesis and angiogenesis in oral cavity and lower lip squamous cell carcinoma / Linfangiogênese e angiogênese em carcinomas de células escamosas de lábio inferior e da cavidade oral

ABSTRACT INTRODUCTION: Tumors of the lip and oral cavity differ in various aspects; therefore a clarification of the distinctions among these sites may help to better understand the biologic behavior of neoplasms occurring in these locations. OBJECTIVE: Considering that angiogenesis and lymphangiogenesis are two major elements that can influence various aspects of tumor biology, we aimed to compare these factors between squamous cell carcinoma of the lower lip and oral cavity. METHODS: A total of 84 primary squamous cell carcinomas including 45 oral and 39 lower lip tumors were selected and immunohistochemically stained with monoclonal antibody against D2-40 and CD105. Mean microvessel density was assessed in tumoral tissue, while lymphatic vessel density was calculated in both neoplastic tissue and invasion front. Data were statistically analyzed using t-test and p-values of <0.05 were considered significant. RESULTS: We found a mean microvessel density ± standard deviation of 31.94 ± 18.9 in oral cavity and 27.54 ± 20.8 in lower lip squamous cell carcinomas, with no significant difference (p = 0.32). Mean lymphatic vessel density ± standard deviation was 13.05 ± 8.2 and 16.57 ± 10.79 in of oral cavity and lower lip neoplastic tissue, respectively. The corresponding values were 9.94 ± 5.59 and 12.50 ± 7.8 in the invasive front. Significant differences were not observed in either of the lymphatic vessel density variables between the two sites. CONCLUSION: According to our results, it seems that the search for additional factors other than those related to the vasculature should continue, to help clarify the differences in biologic behavior between lower lip and oral cavity squamous cell carcinomas.

Resumo Introdução: Os tumores de lábio e da cavidade oral diferem em vários aspectos; portanto, o conhecimento das diferenças entre eles pode ajudar na melhor compreensão do comportamento biológico das neoplasias que ocorrem nesses locais. Objetivo: Considerando que a angiogênese e a linfangiogênese são dois elementos importantes que podem influenciar diversos aspectos da biologia dos tumores, objetivamos comparar esses fatores entre o carcinoma de células escamosas (CCE) de lábio inferior e da cavidade oral. Método: No total, foram selecionados 84 CCEs primários (45 tumores da cavidade oral e 39 tumores de lábio). Esses tumores foram corados por processo imunohistoquímico com anticorpo monoclonal anti-D2-40 e CD105. Avaliamos a densidade média de microvasos (DMV) no tecido tumoral, enquanto que a densidade vascular linfática (DVL) foi calculada tanto no tecido neoplásico como no front de invasão. Os dados foram estatisticamente analisados com o uso do teste t e valores de p < 0,05 foram considerados significantes. Resultados: Chegamos a uma média para DMV ± DP de 31,94 ± 18,9 para CCEs na cavidade oral e de 27,54 ± 20,8 no lábio inferior, sem diferença significante (p = 0,32). As médias para DVL ± DP foram de 13,05 ± 8,2 e 16,57 ± 10,79 no tecido neoplásico da cavidade oral e lábio inferior, respectivamente. Os valores correspondentes foram 9,94 ± 5,59 e 12,50 ± 7,8 no front invasivo. Não foram observadas diferenças significantes nas duas variáveis DVL entre os dois locais. Conclusão: De acordo com os nossos resultados, a pesquisa por fatores adicionais, além daqueles relacionados à vasculatura, deve ter continuidade, para auxiliar no esclarecimento das diferenças do comportamento biológico entre CCEs no lábio inferior e na cavidade oral.

Interaction of stromal and microvascular components in keratocystic odontogenic tumors.

OBJECTIVE: Little is known about the interaction of stromal components in odontogenic tumors. Thus, the aim of this study was to investigate mast cells (MCs), myofibroblasts, macrophages, and their possible association with angiogenesis and lymphangiogenesis in keratocystic odontogenic tumors (KCOTs). MATERIAL AND METHODS: Thirty cases of KCOTs were included and analyzed by immunohistochemistry for mast cell tryptase, α-SMA, CD34, CD163, and D240. For comparative purpose, 15 radicular cysts (CRs) and 7 pericoronal follicles (PFs) were included. RESULTS: There was an increase in MCs for RCs and this difference was significant when they were compared to KCOTS and PFs. A significant increase in the density of MFs was observed for KCOTs when compared to RCs and PFs (P = 0.00). No significant difference in CD163-positive macrophages (P = 0.084) and CD34-positive vessels (P = 0.244) densities was observed between KCOTs, RCs, and PFs, although KCOTs showed a higher density of all proteins. Significant difference in lymphatic vessel density was observed for KCOTs when compared to RCs and PFs (P = 0.00). Positive correlation was observed between mast cell tryptase and CD34 in KCOTs (P = 0.025). CONCLUSIONS: A significant interaction between the MC population and CD34-positive vessels in KCOTs supported the hypothesis that MCs and blood vessels contribute to the stromal scaffold of KCOT.

Lymphangiogenesis and angiogenesis in oral cavity and lower lip squamous cell carcinoma.

INTRODUCTION: Tumors of the lip and oral cavity differ in various aspects; therefore a clarification of the distinctions among these sites may help to better understand the biologic behavior of neoplasms occurring in these locations. OBJECTIVE: Considering that angiogenesis and lymphangiogenesis are two major elements that can influence various aspects of tumor biology, we aimed to compare these factors between squamous cell carcinoma of the lower lip and oral cavity. METHODS: A total of 84 primary squamous cell carcinomas including 45 oral and 39 lower lip tumors were selected and immunohistochemically stained with monoclonal antibody against D2-40 and CD105. Mean microvessel density was assessed in tumoral tissue, while lymphatic vessel density was calculated in both neoplastic tissue and invasion front. Data were statistically analyzed using t-test and p-values of <0.05 were considered significant. RESULTS: We found a mean microvessel density±standard deviation of 31.94±18.9 in oral cavity and 27.54±20.8 in lower lip squamous cell carcinomas, with no significant difference (p=0.32). Mean lymphatic vessel density±standard deviation was 13.05±8.2 and 16.57±10.79 in of oral cavity and lower lip neoplastic tissue, respectively. The corresponding values were 9.94±5.59 and 12.50±7.8 in the invasive front. Significant differences were not observed in either of the lymphatic vessel density variables between the two sites. CONCLUSION: According to our results, it seems that the search for additional factors other than those related to the vasculature should continue, to help clarify the differences in biologic behavior between lower lip and oral cavity squamous cell carcinomas.

Promoting inflammatory lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) aggravated intestinal inflammation in mice with experimental acute colitis

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C), in a mouse model of acute colitis. Forty eight-week-old female C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0±3.9 vs 38.2±1.9, P<0.001) and lymphatic vessel size (1974.6±104.3 vs 1639.0±91.5, P<0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0±1.4 vs 3.5±0.4, P<0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.

A linfangiogênese em enxertos corneanos humanos que evoluem para retransplante / Lymphangiogenesis in human corneal grafts that has evolved to re-transplantation

RESUMO Objetivo: Estudar botões corneanos humanos com linfangiogênese através do exame histopatológico, juntamente com os enxertos de seus transplantes anteriores e posteriores, avaliando os intervalos de tempo para sucessivas cirurgias. Métodos: Estudo descritivo, observacional, longitudinal de botões corneanos humanos com linfangiogênese, juntamente com seus transplantes anteriores e posteriores. Os tecidos foram provenientes de ceratoplastia penetrante no período compreendido entre os anos 2006 e 2013. Após revisão de prontuários em que foram obtidas principalmente as datas das cirurgias, construímos uma tábua de sobrevivência a partir da qual os intervalos de tempo para retransplante foram calculados. Resultados: Entre 89 casos de linfangiogênese corneana, foram incluídos apenas aqueles 22 que possuíam registros no prontuário de transplantes anteriores ou posteriores. Nos casos que apresentavam como provável etiologia do retransplante a linfangiogênese, isolada ou associada à hemangiogênese (grupos pré-linfangiogênese/linfangiogênese e interlinfangiogênese), foram encontrados intervalos de tempo para retransplante menores (7 e 3 meses, respectivamente) que aquele encontrado no grupo linfangiogênese/pós-linfangiogênese que apresentava outras etiologias prováveis para os retransplantes (11,31 meses). Casos que apresentavam como etiologia provável do retransplante a linfangiogênese isolada apresentaram um intervalo para retransplante (3 meses) ainda menor que aquele encontrado nos casos em que a etiologia provável era a linfangiogênese associada à hemangiogênese (7,80 meses). Conclusão: Linfangiogênese, isolada ou associada à hemangiogênese, foi encontrada nos enxertos corneanos humanos estudados que evoluíram para retransplante em pequenos intervalos de tempo. Esse achado nos leva a sugerir um possível papel para os vasos linfáticos na redução do tempo de sobrevida dos enxertos corneanos humanos.

ABSTRACT Objective: To study human corneal buttons with lymphangiogenesis through histopathological examination, together with the grafts of their preceding and subsequent transplantations, evaluating the time intervals for successive surgeries Methods: A descriptive, observational and longitudinal study of human corneal buttons that have lymphatic vessels, together with its preceding and subsequent transplants. Tissues were obtained from penetrating keratoplasty in the period between the years 2006 and 2013. After a medical records review in which information on the dates of the surgeries were mainly obtained, we built a survival table from which the time intervals for retransplantation were calculated. Results: Among 89 cases of corneal lymphangiogenesis, we included only those 22, which had previous or subsequent transplantations records in medical records. In cases where the probable regrafting etiology were lymphangiogenesis, alone or combined with hemangiogenesis (pre-lymphangiogenesis/lymphangiogenesis and interlymphangiogenesis groups), time intervals for retransplantation were found to be minor (7 and 3 months, respectively) than that found in lymphangiogenesis/post-lymphangiogenesis group that had other probable etiologies for retransplantations (11.31 months). Cases that had isolated lymphangiogenesis as probable etiology of retransplantation showed an interval time for retransplantation (3 months) lower than that found in cases in which the probable etiology was lymphangiogenesis associated with hemangiogenesis (7.80 months). Conclusion: Lymphangiogenesis, alone or combined with hemangiogenesis, was found in human corneal grafts studied that have evolved to regraft in small time intervals. This finding leads us to suggest a possible role for the lymphatic vessels in reducing the human corneal grafts survival time.

Serum concentration and immunostaining of vascular endothelial growth factor in dogs with multicentric lymphoma / Concentração sérica e imunomarcação do fator de crescimento do endotélio vascular em cães com linfoma multicêntrico

Vascular endothelium growth factor (VEGF) is one of the most specific regulators of tumor angiogenesis and lymphangiogenesis. Moreover, VEGF plays a role in tumorigenesis through activation of an autocrine signaling pathway that enables cancer cells to stimulate their own growth. The aim of this study was to investigate VEGF serum levels and immunoexpression in canine multicentric lymphomas and correlate these parameters with the prognostic factors of this cancer. Sixteen dogs were evaluated (eight clinically healthy and eight diagnosed with multicentric lymphoma). The animals underwent blood sampling to measure VEGF serum concentrations and lymph node biopsy to evaluate VEGF immunoexpression. The VEGF immunoreactivity score was higher (p=0.0003) in the lymph nodes of dogs with multicentric lymphoma (8.50±2.33) than in those of healthy dogs (1.87±1.80). There was no significant difference in the VEGF serum concentrations between healthy dogs and dogs with lymphoma (p=0.08). Immunophenotype, clinical stage and grade of malignancy influenced the life expectancy of dogs with lymphoma. Our results showed that VEGF is expressed in high amounts in the lymph nodes of dogs with multicentric lymphoma and may be responsible for the growth, survival and migration of tumor cells.

O fator de crescimento do endotélio vascular (VEGF) é um dos mais específicos reguladores da angiogênese e da linfangiogênese tumoral, além de influenciar na tumorigênese mediante ativação de um circuito de sinalização autócrina que permite que as células neoplásicas estimulem seu próprio crescimento. O objetivo desse estudo foi investigar os níveis séricos e a imunomarcação do VEGF nos linfomas multicêntricos caninos e correlacionar esses parâmetros com fatores prognósticos da neoplasia. Foram avaliados 16 cães, sendo oito clinicamente sadios e oito com diagnóstico de linfoma multicêntrico. Os animais foram submetidos à coleta de sangue para mensuração da concentração sérica do VEGF e biopsia de linfonodo para avaliação da imunomarcação do VEGF. O escore de imunomarcação do VEGF foi superior (p=0,0003) nos linfonodos de cães com linfoma multicêntrico (8,50±2,33) em comparação ao escore dos linfonodos de cães sadios (1,87±1,80). Não houve diferença significativa entre as concentrações séricas do VEGF de cães sadios e de cães com linfoma (p=0,08). Imunofenótipo, estadiamento clínico e grau de malignidade influenciaram na sobrevida dos cães com linfoma. Os resultados obtidos permitiram concluir que o VEGF está expresso em quantidades elevadas nos linfonodos de cães com linfoma multicêntrico, podendo ser responsável pelo crescimento, sobrevivência e migração das células tumorais.(AU)

The main findings in histopathological examination of human corneal buttons with lymphangiogenesis / Principais achados do exame histopatológico de botões corneanos humanos com linfangiogênese

Objective: To detect the presence of lymphatic vessels in the cornea through histopathological examination, trying to identify findings that are most commonly found with the presence of these vessels in this tissue. Methods: Retrospective descriptive study of human corneal buttons with lymphangiogenesis. Tissues were obtained from penetrating keratoplasty in the period between the years 2006 and 2013. A medical record review was conducted looking for information about sex, age and graft etiology. Results: 89 corneal buttons were included, out of which 37 were from female patients and 52 were from male patients. The average age was 47.70 ± 23.95 years (mean ± SD). Lymphangiogenesis was found mainly associated with hemangiogenesis. However, isolated lymphangiogenesis was observed in 28 (31.46%) patients. In 18 (20.22%) cases were found an amount of lymphatic vessels approximately four times higher than that found in most part of the sample. A lot of cases were found in inflammatory conditions such as infection and perforation. Near the lymphangiogenesis, we found many cases of anterior synechia and myofibroblasts. In 17 (16.35%) cases, no change was observed in the vicinity of corneal lymphatic vessels. Conclusions: We demonstrated through a histopathological examination, that findings admittedly associated with lymphangiogenesis like inflamamatory processes, are also frequently found in cases of human corneas that have lymphatic vessels. However, other findings such as lymphangiogenesis without the presence of angiogenesis, the presence of a greater amount of vessels in some cases and lymphangiogenesis without changes in its proximity remain in need of a better understanding.

Objetivo: Detectar a presença de vasos linfáticos na córnea através do exame histopatológico, buscando identificar os achados que são encontrados com maior frequência junto à presença desses vasos nesse tecido. Métodos: Estudo retrospectivo descritivo de botões corneanos humanos com linfangiogênese. Os tecidos foram provenientes de ceratoplastia penetrante no período compreendido entre os anos de 2006 e 2013. Foi realizada revisão de prontuários em busca de informações sobre sexo, idade e etiologia do transplante. Resultados: Foram incluídos 89 botões corneanos, sendo 37 de pacientes do sexo feminino e 52 do sexo masculino. A média das idades foi de 47,70 ± 23,95 anos (média ± DP). Linfangiogênese foi encontrada principalmente associada à hemangiogênese. Linfangiogênese isolada, no entanto, foi observada em 28 (31,46%) casos. Em 18 (20,22%) casos foram encontrados uma quantidade de vasos linfáticos cerca de quatro vezes maior que aquela encontrada na maioria das amostras. Em um grande número de casos foram encontrados condições inflamatórias como infecção e perfuração. Nas proximidades da linfangiogênese, encontramos muitos casos de sinéquia anterior e miofibroblastos. Em 17 (16,35%) casos nenhuma alteração foi evidenciada nas proximidades dos vasos linfáticos corneanos. Conclusão: Demonstramos, através do exame histopatológico, que achados reconhecidamente associados à linfangiogênese, como os processos inflamatórios, são encontrados também com frequência em casos de córneas humanas que possuem vasos linfáticos. Porém, outros achados evidenciados, como a linfangiogênese desacompanhada de angiogênese, a presença de uma maior quantidade de vasos em alguns casos e a linfangiogênese sem alterações em sua proximidade, permanecem necessitando de uma melhor compreensão.

Head and neck squamous cell carcinoma lymphatic spread and survival: Relevance of vascular endothelial growth factor family for tumor evaluation.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is primarily a locoregional disease in which the cervical lymph nodes are the chief site of metastasis. The purpose of this study was to examine the relationship between lymphangiogenesis and clinicopathological aspects of HNSCC and its metastasis. METHODS: Fifty-two patients with HNSCC and metastatic lymph nodes from 21 of these subjects were analyzed by immunohistochemistry. RESULTS: The HNSCC samples were predominantly negative for vascular endothelial growth factor (VEGF)-C, VEGF-D, and vascular endothelial growth factor receptor (VEGFR)3. There was an association between the density of lymph vessels (measured by D2-40 staining) in the lymph nodes and advanced-stage tumors. There was no link between the expression of these proteins and survival rates. CONCLUSION: Although lymphatic spread is a significant event in the progression of HNSCC, the expression of VEGF-C, VEGF-D, and VEGFR3 does not correlate with clinicopathological characteristics, suggesting that other signaling pathways mediate lymphangiogenesis in HNSCC.

Serum concentration and immunostaining of vascular endothelial growth factor in dogs with multicentric lymphoma / Concentração sérica e imunomarcação do fator de crescimento do endotélio vascular em cães com linfoma multicêntrico

Vascular endothelium growth factor (VEGF) is one of the most specific regulators of tumor angiogenesis and lymphangiogenesis. Moreover, VEGF plays a role in tumorigenesis through activation of an autocrine signaling pathway that enables cancer cells to stimulate their own growth. The aim of this study was to investigate VEGF serum levels and immunoexpression in canine multicentric lymphomas and correlate these parameters with the prognostic factors of this cancer. Sixteen dogs were evaluated (eight clinically healthy and eight diagnosed with multicentric lymphoma). The animals underwent blood sampling to measure VEGF serum concentrations and lymph node biopsy to evaluate VEGF immunoexpression. The VEGF immunoreactivity score was higher (p=0.0003) in the lymph nodes of dogs with multicentric lymphoma (8.50±2.33) than in those of healthy dogs (1.87±1.80). There was no significant difference in the VEGF serum concentrations between healthy dogs and dogs with lymphoma (p=0.08). Immunophenotype, clinical stage and grade of malignancy influenced the life expectancy of dogs with lymphoma. Our results showed that VEGF is expressed in high amounts in the lymph nodes of dogs with multicentric lymphoma and may be responsible for the growth, survival and migration of tumor cells.

O fator de crescimento do endotélio vascular (VEGF) é um dos mais específicos reguladores da angiogênese e da linfangiogênese tumoral, além de influenciar na tumorigênese mediante ativação de um circuito de sinalização autócrina que permite que as células neoplásicas estimulem seu próprio crescimento. O objetivo desse estudo foi investigar os níveis séricos e a imunomarcação do VEGF nos linfomas multicêntricos caninos e correlacionar esses parâmetros com fatores prognósticos da neoplasia. Foram avaliados 16 cães, sendo oito clinicamente sadios e oito com diagnóstico de linfoma multicêntrico. Os animais foram submetidos à coleta de sangue para mensuração da concentração sérica do VEGF e biopsia de linfonodo para avaliação da imunomarcação do VEGF. O escore de imunomarcação do VEGF foi superior (p=0,0003) nos linfonodos de cães com linfoma multicêntrico (8,50±2,33) em comparação ao escore dos linfonodos de cães sadios (1,87±1,80). Não houve diferença significativa entre as concentrações séricas do VEGF de cães sadios e de cães com linfoma (p=0,08). Imunofenótipo, estadiamento clínico e grau de malignidade influenciaram na sobrevida dos cães com linfoma. Os resultados obtidos permitiram concluir que o VEGF está expresso em quantidades elevadas nos linfonodos de cães com linfoma multicêntrico, podendo ser responsável pelo crescimento, sobrevivência e migração das células tumorais.

Expression of lymphatic markers and lymphatic growth factors in psoriasis before and after anti-TNF treatment

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results. .

FASN expression, angiogenesis and lymphangiogenesis in central and peripheral giant cell lesions

Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) are non-neoplastic proliferative processes of the jaws. PGCL is a reactive process induced by irritant local factors and CGCL is an intra-osseous lesion of unknown etiology. Both lesions exhibit similar histologic features showing abundant mononuclear cells, admixed with a large number of multinucleated giant cells and a rich vascularized stroma with extravasated erythrocytes, hemosiderin deposition, and blood-filled pools. Recent studies have linked fatty acid synthase (FASN) with angiogenesis. Objective: To evaluate angiogenesis and lymphangiogenesis and their relationship with FASN expression in CGCL and PGCL. Material and Methods: Thirteen CGCL and 14 PGCL of the jaws were selected for immunoexpression of FASN; CD34 and CD105 (to assess blood microvessel density [MVD] and microvessel area [MVA]); and D2-40 (to assess lymphatic MVD and MVA). Results: Within PGCL and CGCL, MVD-CD34 was signifcantly higher than MVD-CD10S, followed by MVD-D2-40. Moreover, a signifcantly higher number of FASN-positive multinucleated giant cells than mononuclear cells were observed. Between PGCL and CGCL, only MVD-CD34 and all MVA were signifcantly higher in PGCL. Positive correlation between MVA-CD10S with FASNpositive mononuclear cells in both lesions was observed. Conclusions: Our results show both lesions exhibiting similar levels of FASN expression and neoangiogenesis, suggesting constitutive processes that regulate tissue maintenance. .