Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing

Vascular endothelial growth factor receptor-3 (VEGFR-3) is essential for embryonic cardiovascular development, but thereafter becomes confined to the lymphatic endothelium in adult tissues. We have here studied VEGFR-3 expression in experimental wounds of pigs and chronic inflammatory wounds of humans. In healing incisional and punch biopsy wounds made in the dorsal skin of pigs, angiogenic blood vessels, identified by use of the blood vascular endothelial markers vWF and PAL-E and the basal lamina protein laminin, developed into the granulation tissue stroma from day 4 onward, being most abundant on days 5 and 6 and regressing thereafter. VEGFR-3-positive vessels were observed in the granulation tissue from day 5 onward. These vessels were distinct from the PAL-E/laminin/vWF-positive vessels and fewer in number, and they appeared to sprout from pre-existing VEGFR-3-positive lymphatic vessels at the wound edge. Unlike the blood vessels, very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. In chronic wounds such as ulcers and decubitus wounds of the lower extremity of humans, VEGFR-3 was also weakly expressed in the vascular endothelium. Our results suggest that transient lymphangiogenesis occurs in parallel with angiogenesis in healing wounds and that VEGFR-3 becomes up-regulated in blood vessel endothelium in chronic inflammatory wounds.     

Platelet-derived growth factors induced lymphangiogenesis: evidence,unanswered questions and upcoming challenges

Crosstalk between angiogenesis and lymphangiogenesis in embryonic development continues during postnatal life and has specific mechanisms involving factors that initiate activation of the intracellular cascade for their specific receptors. Platelet-derived growth factors (PDGFs) and their corresponding receptors (PDGFRs) are known as important regulators of blood vessel development in both normal and pathologic angiogenesis. Despite some recent papers which reported a potential role of the PDGF/PDGFR axis in lymphatic spread of tumor cells, a few papers have suggested the potential role of PDGFs in tumor lymphangiogenesis development. The present paper summarizes the potential lymphangiogenic role of the PDGF/PDGFR axis, underlying upcoming challenges in the field.     

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.     

Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas.

Capillary hemangioblastomas and hemangiopericytomas are highly vascular central nervous system tumors of controversial origin. Of interest in their pathogenesis are mechanisms regulating endothelial cell growth. The endothelial cell mitogen vascular endothelial growth factor (VEGF) stimulates angiogenesis, and together with its two receptor tyrosine kinases VEGFR-1(FLT1) and VEGFR-2(KDR), is up-regulated during the malignant progression of gliomas. We have analyzed the expression of VEGF and its receptors, the related placental growth factor (PlGF) and the endothelial receptors FLT4 and Tie by in situ hybridization in capillary hemangioblastomas and hemangiopericytomas. VEGF mRNA was up-regulated in all of the hemangiopericytomas studied and highly expressed in the stromal cells of hemangioblastomas. In addition, some hemangioblastoma tumor cells expressed high levels of PlGF. Significantly elevated levels of Tie mRNA, Tie protein, VEGFR-1, and VEGFR-2 but not FLT4 mRNAs were observed in the endothelia of both tumor types. In hemangioblastomas, however, the receptors were also highly expressed by a subpopulation of stromal cells. Consistent results were obtained for a human hemangioblastoma cell line in culture. Up-regulation of the endothelial growth factors and receptors may result in autocrine or paracrine stimulation of endothelial cells and their precursors involved in the genesis of these two vascular tumors.     

Vascular endothelial growth factors and angiopoietins as new players in mastocytosis

Clinical and Experimental Medicine - Mastocytosis is a disorder characterized by the abnormal proliferation and/or accumulation of mast cells in different organs. More than 90% of patients with...     

Stromal impact on tumor growth and lymphangiogenesis in human carcinoma xenografts

Squamous cell carcinomas (SCCs) arising in the oral cavity are associated with poor survival, mainly due to metastatic disease. In contrast, skin SCCs rarely metastasize and are usually curable. To study influence of tongue and skin stroma on cancer growth and induction of lymphangiogenesis, xenograft tumors of human carcinoma cells were established either in tongue or skin of BALB/c nude mice. Two oral and two skin SCC cell lines were used, as well as an endometrial adenocarcinoma cell line. Tongue tumors established from all cell lines were larger than corresponding skin tumors. Peritumoral lymphatic vessel density was up to five times higher in tongue than in corresponding skin tumors, and mRNA level of the lymphangiogenic growth factor vascular endothelial growth factor (VEGF)-C was twice as high in tongue tumors compared with corresponding skin tumors. Contrary to lymphatic vessel density, blood vessel density was higher in skin tumors than in tongue tumors. In a cohort of patient samples, lymphatic vessel density was found to be higher in tongue SCCs compared with skin SCCs, supporting a clinical relevance of our findings. Our results show that the tumor stroma has a profound impact on cancer growth and induction of lymphangiogenesis and angiogenesis. The difference in lymphatic vessel density between tongue and skin tumors may be important in directing metastatic potential of tumors arising in these organs.     

Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis

In many human carcinomas, expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) correlates with up-regulated lymphangiogenesis and regional lymph node metastasis. Here, we have used the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis to investigate the functional role of VEGF-D in the induction of lymphangiogenesis and tumor progression. Expression of VEGF-D in beta cells of single-transgenic Rip1VEGF-D mice resulted in the formation of peri-insular lymphatic lacunae, often containing leukocyte accumulations and blood hemorrhages. When these mice were crossed to Rip1Tag2 mice, VEGF-D-expressing tumors also exhibited peritumoral lymphangiogenesis with lymphocyte accumulations and hemorrhages, and they frequently developed lymph node and lung metastases. Notably, tumor outgrowth and blood microvessel density were significantly reduced in VEGF-D-expressing tumors. Our results demonstrate that VEGF-D induces lymphangiogenesis, promotes metastasis to lymph nodes and lungs, and yet represses hemangiogenesis and tumor outgrowth. Because a comparable transgenic expression of vascular endothelial growth factor-C (VEGF-C) in Rip1Tag2 has been shown previously to provoke lymphangiogenesis and lymph node metastasis in the absence of any distant metastasis, leukocyte infiltration, or angiogenesis-suppressing effects, these results reveal further functional differences between VEGF-D and VEGF-C.     

Vascular genetic factors and human longevity

Complex inter-relationships between age-associated illnesses, such as vascular disease and Alzheimer's disease (AD), suggest that biological and genetic pathways may be worthy of examination in centenarian populations to provide insights into human longevity. This is also borne out by the involvement of lipoprotein metabolism and a number of vascular genetic risk factors. Repeated findings of a higher frequency of the apolipoprotein E (APOE) epsilon4 allele in middle-aged subjects compared with centenarians were reported. Furthermore, we have also shown how in different populations there is a significant trend in reduction of serum APOE levels from APOE epsilon2- to epsilon4-carrier as well as significant differences in serum APOE levels respect to age in epsilon4-carriers but only after adjustment for HDL cholesterol. In contrast, findings of increased prevalence of the angiotensin I converting enzyme 1 (ACE1) D allele in French centenarians have not been replicated, suggesting the possibility that regional differences may occur in ACE1(*)D frequency within Europe in centenarians, as has been recently reported for APOE epsilon2 and epsilon4 alleles. A number of studies have examined the potential role in longevity of other genes involved in vascular risk, haemostasis, and blood pressure regulation [methyltetrahydrofolatereductase (MTHFR), apolipoprotein A1 (APOA-I), apolipoprotein C3 (APOC-III), apolipoprotein A4 (APOA-IV), paraoxonase 1 (PON1), plasminogen activator inhibitor type I (PAI-1)], with contrasting results. While further studies are needed to confirm the possible role of APOE concentration as putative longevity factor, this paper provides an overview of genetic vascular factors potentially involved in human longevity.     

Vascular factors altered in glucose-treated mesangial cells and diabetic glomeruli. Changes in vascular factors impair endothelial cell growth and matrix

We studied the effect of a high glucose (HG) environment on the vascular factors that are secreted by mesangial cells, and regulate endothelial growth and mesangial matrix deposition. To this effect, we measured the vascular factors in the glomeruli of streptozotocin-induced diabetic kidneys and in mesangial cells exposed to a HG concentration. We then transferred the media of mesangial cells previously exposed to high glucose to cultured endothelial cells to study the effects on endothelial growth, matrix formation, and in vitro capillary proliferation. In 1-week diabetic kidneys, glomerular vascular endothelial growth factor (VEGF) and angiopoietin-1 were inhibited by 38 and 57%, respectively, but angiopoietin-2 was increased by 318%. We found similar results in mesangial cells exposed to HG. There was a decrease of VEGF (50% by enzyme immunoassay, 27% by mRNA), decrease of angiopoietin-1 (65% by mRNA), and a much greater increase of angiopoietin-2 (280% by immunoassay, 523% by mRNA). Compared to controls, the media of mesangial cells previously exposed to HG impaired endothelial cell growth by 61%, increased extracellular matrix by 100%, and decreased capillary formation by 90%. We conclude that high ambient glucose alters the secretion of vascular factors elaborated by mesangial cells, resulting in an expansion of the endothelial cell matrix and disruption of capillary structure.     

Intratumoral lymphangiogenesis of esophageal squamous cell carcinoma and relationship with regulatory factors and prognosis

The clinical and pathological significance of intratumoral lymphangiogenesis (ITL) with human esophageal squamous cell carcinomas (ESCC) remains unclear, as does the role of signaling molecules such as vascular endothelial growth factor (VEGF)-A,C, platelet-derived growth factor (PDGF)-A, and p53, in the regulation of ITL. Lymphatic vessel density (LVD) was significantly increased in VEGF-A and VEGF-C immunohistochemical score 1 and 2-3 groups as compared to the score 0 group and also with high of VEGF-A, VEGF-C and PDGF-A mRNA expression. Both LVD and blood vessel density (BVD) were significantly greater in the p53 gene mutant group than in the wild-type group. Lymph node metastasis was significantly more frequent with than without ITL and Kaplan-Meier analysis indicated a significantly poorer prognosis. Multivariate analysis using Cox proportional hazard method showed that invasion depth, lymph node metastasis and ITL were independent prognostic factors.     

吲哚类化合物MAZ51对小鼠移植瘤生长及淋巴管生成的影响

目的:观察吲哚类化合物MAZ51对小鼠移植瘤生长及淋巴管生成的影响,并探讨其作用机制.方法:皮下注射肉瘤(S180)腹水型瘤株构建小鼠移植瘤模型,成瘤后给予MAZ51,观察其对移植瘤生长的影响.免疫组织化学标记淋巴管,分析移植瘤周边区和正常皮肤的毛细淋巴管密度(LMVD).结果:移植瘤周边区的LMVD高于正常皮肤组织.给予MAZ51后,明显减慢移植瘤的生长,但瘤周边区的LMVD并没有变化.结论:实验结果显示肿瘤周边区有淋巴管新生.MAZ51影响移植瘤的生长,但不影响淋巴管的生成.     

Vascular cells respond differentially to transforming growth factors beta 1 and beta 2 in vitro.

Transforming growth factor beta 1 (TGF-beta 1) and beta 2 (TGF-beta 2) are equipotent in many cell systems studies thus far. Recent data, however, show different effects elicited by these two growth factors in specific biologic systems. This investigation compares the effects of TGF-beta 1 and TGF-beta 2 bovine aortic endothelial cells (BAECs), rat epididymal fat pad microvascular endothelium (RFCs), and bovine aortic smooth muscle cells (BASCs). In two-dimensional cultures, proliferation of BAECs, BASMCs, and RFCs were all inhibited by TGF-beta 1, while in response to TGF-beta 2, BASMCs were fully inhibited, RFCs were modestly inhibited, and BAECs were unaffected. Bovine aortic endothelial cell migration was significantly inhibited by TGF-beta 1, but only slightly inhibited by TGF-beta 2. In contrast, BASMC migration was enhanced by TGF-beta 1 and was not affected by TGF-beta 2. In three-dimensional cultures, RFCs were stimulated to undergo in vitro angiogenesis in response to TGF-beta 1 and TGF-beta 2 at 10-fold higher concentrations. Three distinct receptor assays demonstrated the presence of type I and type II TGF-beta 1 cell-surface-binding proteins on BAECs, BASMCs, and RFCs. Labeled TGF-beta 1 was competed off completely with 100-fold molar excess unlabeled TGF-beta 1, but only partially with equivalent excess unlabeled TGF-beta 2. Furthermore the ratios of type I to type II TGF-beta receptors in these three vascular cell types vary from 1:1 in BAECs to 1.5:1 in RFCs to 3:1 in BASMCs and can be correlated with the differences noted in cellular responses to TGF-beta 1 and TGF-beta 2 in proliferation, migration, and in vitro angiogenic assays. These findings support the hypothesis that there are different responses to the TGF-beta s, depending on the cell type and experimental conditions as well as the TGF-beta concentration and isoform used.     

细胞因子与肿瘤淋巴管形成

在很多恶性肿瘤中,淋巴道转移是其主要的转移途径,其在肿瘤的诊断、分期和治疗中起着关键的作用.肿瘤诱导血管形成对于肿瘤生长和转移的意义已经在研究者之间达成共识.研究发现,与血管生成类似,淋巴管形成参与胞外基质退化,刺激淋巴内皮细胞增殖和迁移,以及促进管状结构的形成[1].能在细胞间传递信息和具有免疫调节的的细胞因子自然在其形成中也起到重要作用.临床上已经利用包括是肿瘤分泌的和宿主细胞分泌的细胞因子的作用机制研制药物并治疗疾病.细胞因子在淋巴管形成中的作用机制会具有更广阔的价值.     

Vascular endothelial growth factor and endometriotic angiogenesis

Peritoneal endometriosis is a significant debilitating gynaecological problem of widespread prevalence. It is now generally accepted that the pathogenesis of peritoneal endometriosis involves the implantation of exfoliated endometrium. Essential for its survival is the generation and maintenance of an extensive blood supply both within and surrounding the ectopic tissue. The vascular endothelial growth factor (VEGF) family of angiogenic molecules is involved in both physiological angiogenesis, and a number of pathological conditions that are characterized by excessive angiogenesis. Increasing evidence suggests that the VEGF family may also be involved with both the aetiology and maintenance of peritoneal endometriosis. Sources of this factor include the eutopic endometrium, ectopic endometriotic tissue and peritoneal fluid macrophages. Important to its aetiology is the correct peritoneal environment in which the exfoliated endometrium is seeded and implants. Established ectopic tissue is then dependent on the peritoneal environment for its survival, an environment that supports angiogenesis. Our increasing knowledge of the involvement of the VEGF family in endometriotic angiogenesis raises the possibility of novel approaches to its medical management, with particular focus on the anti-angiogenic control of the action of VEGF.     

塞来昔布对小鼠移植瘤生长及淋巴管生成的影响

目的观察塞来昔布(Celecoxib)对小鼠移植瘤生长、COX-2、VEGF-C表达和淋巴管生成影响,探讨抑制肿瘤淋巴转移的机制。方法构建S180小鼠移植瘤模型,应用大体观察、HE染色、免疫组化和Western blot,比较对照组和Celecoxib给药组荷瘤鼠肿瘤生长、COX-2、VEGF-C表达和淋巴管分布。结果 Celecoxib组与对照组相比瘤体生长缓慢,第8周时肿瘤体积明显比对照组小,COX-2和VEGF-C的表达均下调,淋巴管密度降低。结论 Celecoxib抑制肿瘤的转移和生长可能与下调COX-2表达,减少VEGF-C的产生和淋巴管生成有关。     

Multiplexed analysis of angiogenesis and lymphangiogenesis factors predicts outcome for non-small cell lung cancer patients

Angiogenesis and lymphangiogenesis are key components of non-small cell lung cancer (NSCLC) tumor growth and metastatic spread; however, the prognostic and predictive role of angiogenic and lymphangiogenic biomarkers remains controversial for NSCLC patients. We assessed VEGF, VEGFC, VEGFD, VEGFR3 protein expression, tumor microvessel, and lymphatic vessel (LmVD) density by immunohistochemistry in 103 NSCLC; biomarkers were analyzed individually as well as multiplexed with each other. No correlations were identified between VEGF, VEGFC, VEGFD, or LmVD and clinical characteristics. VEGFR3 was correlated with VEGFC (p = 0.03), VEGFD (p < 0.0001), and intratumor LmVD (p = 0.03). Tumors that did not express VEGFR3 had a worse prognosis (log rank p = 0.03). VEGF was significantly correlated with survival in adenocarcinomas (log rank p = 0.014) but not in squamous cell carcinomas (log rank p = 0.5). Multivariate Cox regression analysis confirmed the independent prognostic potential of VEGFR3 (hazard ratio (HR) = 0.05; 95% confidence intervals (CI) = 0.008–0.32, p = 0.002) for all patients and VEGF (HR = 8.69, 95% CI = 1.4–53.69, p = 0.02) for adenocarcinomas. When biomarkers were multiplexed, only stage and VEGFC expression were independent predictors of survival for all patients. Weighted expression of VEGFC, VEGFR3, and stage was used to build a prognostic classifier for stage I–IIIA patients; patients in the low risk group had prolonged survival compared with high risk patients (log rank p = 0.02). There was no association between biomarkers and early recurrence or response to treatment. Angiogenic and lymphangiogenic biomarkers studied define subgroups of patients at high risk and may be useful for prognostic stratification of NSCLC patients especially those with early stage disease.