Regulation of microvascular permeability by vascular endothelial growth factors

Generation of new blood vessels from pre-existing vasculature (angiogenesis) is accompanied in almost all states by increased vascular permeability. This is true in physiological as well as pathological angiogenesis, but is more marked during disease states. Physiological angiogenesis occurs during tissue growth and repair in adult tissues, as well as during development. Pathological angiogenesis is seen in a wide variety of diseases, which include all the major causes of mortality in the west: heart disease, cancer, stroke, vascular disease and diabetes. Angiogenesis is regulated by vascular growth factors, particularly the vascular endothelial growth factor family of proteins (VEGF). These act on two specific receptors in the vascular system (VEGF-R1 and 2) to stimulate new vessel growth. VEGFs also directly stimulate increased vascular permeability to water and large-molecular-weight proteins. We have shown that VEGFs increase vascular permeability in mesenteric microvessels by stimulation of tyrosine auto-phosphorylation of VEGF-R2 on endothelial cells, and subsequent activation of phospholipase C (PLC). This in turn causes increased production of diacylglycerol (DAG) that results in influx of calcium across the plasma membrane through store-independent cation channels. We have proposed that this influx is through DAG-mediated TRP channels. It is not known how this results in increased vascular permeability in endothelial cells in vivo. It has been shown, however, that VEGF can stimulate formation of a variety of pathways through the endothelial cell, including transcellular gaps, vesiculovacuolar organelle formation, and fenestrations. A hypothesis is outlined that suggests that these all may be part of the same process.     

The vascular endothelial growth factor family; proteins which guide the development of the vasculature

The development of the vascular tree during embryogenesis involves vasculogenesis, angiogenesis and tissue-specific differentiation of endothelium which gives rise to many different vessel types. These processes are physiologically complex and are therefore difficult to study in vitro. However, the discovery of endothelial cell-specific receptors and cognate ligands has led to the generation of transgenic and knockout mouse models which have shed light on the molecular mechanisms that regulate the development of blood and lymphatic vessels during embryogenesis. Such mouse models have demonstrated that members of the vascular endothelial growth factor (VEGF) family of proteins and the VEGF receptors are critical regulators of vasculogenesis, angiogenesis and endothelial cell differentiation. The availability of purified VEGF family members and of inhibitors of these growth factors may provide a means to modulate blood vessel growth for the treatment of cancer, retinopathies and diseases of ischemia.     

The role of angiopoietins during angiogenesis in gliomas

The formation of new blood vessels plays an important role in human disease development and progression. For instance, it is well established that the growth of most cancers critically depends on the supply of nutrition and oxygen by newly recruited blood vessels. Similarly, malignant gliomas, the most common primary brain tumors occurring in humans are highly dependent on angiogenesis. In recent years, there has been tremendous effort to uncover the molecular mechanisms that drive blood vessel growth in adult tissues, especially during cancer progression. Vascular endothelial growth factor (VEGF) and other morphogens, such as angiopoietins and ephrins have been shown to be critically involved in the formation of new blood vessels during both developmental and pathological angiogenesis as evidenced by genetic studies in mice. In this review, we focus on angiopoietins, a family of growth factor ligands binding to Tie tyrosine kinase receptors with emphasis on their functional consequences during the growth and progression of experimental tumors and malignant human gliomas.     

Vascular endothelial growth factor-D: signaling mechanisms,biology, and clinical relevance

Vascular endothelial growth factor-D (VEGF-D) is a secreted glycoprotein that promotes growth of blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis), and can induce remodeling of large lymphatics. VEGF-D enhances solid tumor growth and metastatic spread in animal models of cancer, and in some human cancers VEGF-D correlates with metastatic spread, poor patient outcome, and, potentially, with resistance to anti-angiogenic drugs. Hence, VEGF-D signaling is a potential target for novel anti-cancer therapeutics designed to enhance anti-angiogenic approaches and to restrict metastasis. In the cardiovascular system, delivery of VEGF-D in animal models enhanced angiogenesis and tissue perfusion, findings which have led to a range of clinical trials testing this protein for therapeutic angiogenesis in cardiovascular diseases. Despite these experimental and clinical developments, our knowledge of the signaling mechanisms driven by VEGF-D is still evolving—here we explore the biology of VEGF-D, its signaling mechanisms, and the clinical relevance of this growth factor.     

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加     

B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis

Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitt's lymphoma (E micro -c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) E micro -c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in E micro -c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from E micro -c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively.     

Intratumoral Lymphatics and Lymphatic Vessel Invasion Detected by D2‐40 Are Essential for Lymph Node Metastasis in Bladder Transitional Cell Carcinoma

Bladder cancer is frequently associated with regional lymph node metastasis at the time of diagnosis or after initial treatment, and lymph node metastasis is crucial for clinical therapeutic strategies. Lymphangiogenesis, detected by antibodies specific for lymphatic endothelial cells, is correlated with cancer spread, but the mechanisms that underlie lymphatic spread and the role of lymphangiogenesis in cancer metastasis has been less clear. The aim of this study was to investigate the association of vascular endothelial growth factor (VEGF)‐D expression, intratumoral lymphatics, and lymphatic invasion associated with lymph node metastasis as well as the prognostic analysis in patients with bladder transitional cell carcinoma (TCC). The VEGF‐D expression was evaluated by immunohistochemistry in 72 specimens, and tumoral lymphatic vessels were measured by D2‐40. Counts of lymph vessels were taken in intratumoral and peritumoral areas. Survival analyses and their independent roles were investigated using univariate and multivariate analysis models. The high expression of VEGF‐D was closely associated with the intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Higher lymphatic vessel density, intratumoral lymphatics, and lymphatic invasion showed a significant association with lymph node metastasis. Univariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were associated with overall survival, but they were not independent prognostic factors for bladder TCC in multivariate analysis. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis. Intratumoral lymphatics and lymphatic invasion are important predictive factors of pelvic lymph node metastasis in patients with bladder cancer. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

The role of VEGF in normal and neoplastic hematopoiesis

VEGF is a secreted growth factor that mediates its biological effects by binding to two transmembrane tyrosine kinase receptors, VEGFR-1 and VEGFR-2. The VEGF/receptor signaling system is involved in the regulation of two fundamental processes in vertebrates: the formation of blood vessels (angiogenesis) and of blood cells (hematopoiesis). Hematopoietic stem cells, capable of giving rise to all blood cell lineages, are often found in clusters with endothelial cells, the key cell type involved in the formation of blood vessels. Despite such proximity of VEGF-responsive cells, hematopoiesis occurs independently of neoangiogenesis in the adult bone marrow, suggesting that VEGF regulates the two processes by different mechanisms. In support of this hypothesis, the recently identified autocrine loop by which VEGF may control hematopoietic stem cell survival and repopulation, is fundamentally different from its paracrine effects regulating angiogenesis. Furthermore, coexpression of VEGF and its receptors, the prerequisite for autocrine loops, is frequently found in lymphomas and myelomas, suggesting that autocrine loops also play a role in hematological malignancies. Several therapeutic strategies blocking VEGF or VEGF-induced signaling are currently being investigated for the treatment of neoplastic diseases. They differ in their potential to interfere with the autocrine or paracrine effector functions of VEGF during angiogenesis, hematopoiesis, and tumor cell proliferation, properties which may ultimately determine their therapeutic potential.     

Angiogenesis and lymphangiogenesis and expression of lymphangiogenic factors in the atherosclerotic intima of human coronary arteries

Little information regarding the development of lymphangiogenesis in coronary atherosclerosis is available. We immunohistochemically investigated the correlation among intimal neovascularization (CD34 for angiogenesis and lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin for lymphangiogenesis), the expression of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGF-D), and the progression of atherosclerosis using 169 sections of human coronary arteries from 23 autopsy cases. The more the atherosclerosis advanced, the more often the neointimas contained newly formed blood vessels ( P < .0001). Vascular endothelial growth factor-C was expressed mostly in foamy macrophages and in some smooth muscle cells, whereas VEGF-D was abundantly expressed in both. The number of VEGF-C-expressing cells, but not that of VEGF-D-expressing cells, was increased as the lesion advanced and the number of intimal blood vessels increased ( P < .01). Lymphatic vessels were rare in the atherosclerotic intima (LYVE-1 vs CD34 = 13 vs 3955 vessels) compared with the number seen in the adventitia (LYVE-1 vs CD34 = 360 vs 6921 vessels). The current study suggests that VEGF-C, but not VEGF-D, may contribute to plaque progression and be a regulator for angiogenesis rather than lymphangiogenesis in coronary atherosclerotic intimas. Imbalance of angiogenesis and lymphangiogenesis may be a factor contributing to sustained inflammatory reaction during human coronary atherogenesis.     

Mechanisms of lymphatic metastasis in human colorectal adenocarcinoma

The invasion of lymphatic vessels by colorectal cancer (CRC) and its subsequent spread to draining lymph nodes is a key determinant of prognosis in this common and frequently fatal malignancy. Although tumoural lymphangiogenesis is assumed to contribute to this process, review of the current literature fails to support any notion of a simple correlation between lymphatic vessel density and CRC metastasis. Furthermore, attempts to correlate the expression of various lymphangiogenic growth factors, most notably VEGF‐C and VEGF‐D, with the lymphatic metastasis of CRC have provided contradictory results. Recent evidence from animal and human models of tumour metastasis suggests that complex functional and biochemical interactions between the microvasculature of tumours and other cell types within the tumour microenvironment may play a pivotal role in the behaviour of commonly metastasizing tumours. Indeed, previous insights into tumoural blood vessels have provided candidate markers of tumoural angiogenesis that are currently the subject of intense investigation as future therapeutic targets. In this review article we survey the current evidence relating lymphangiogenesis and lymphangiogenic growth factor production to metastasis by CRC, and attempt to provide some insight into the apparent discrepancies within the literature. In particular, we also discuss some new and provocative insights into the properties of tumoural lymphatics suggesting that they have specific expression profiles distinct from those of normal lymphatic vessels and that appear to promote metastasis. These findings raise the exciting prospect of future biomarkers of lymphatic metastasis and identify potential targets for new generation anti‐tumour therapies. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Control of angiogenesis by VEGF and endostatin-encapsulated protein microcrystals and inhibition of tumor angiogenesis

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals derived from insect cypovirus with overexpression of protein disulfide bond isomerase. VEGF microcrystals were observed to increase the phosphorylation of p42/p44 MAP kinase and to stimulate the proliferation, migration, and network and tube formation of human umbilical vein endothelial cells (HUVECs). Endostatin was also successfully encapsulated into microcrystals. Endostatin microcrystals showed antiangiogenesis activities and inhibited the migration, and network and tube formation of HUVECs. Local administration of endostatin microcrystals in mice inhibited both angiogenesis and tumor growth with clear significant differences between treatment and control groups. Endostatin microcrystals only affected angiogenesis, but had no significant effect on lymphangiogenesis compared to controls. Local therapy using endostatin microcrystals offers a potential approach to achieve sustained therapeutic release of antiangiogenic molecules for cancer treatment.     

Review of the effects of anti-angiogenic compounds on the epiphyseal growth plate

The formation of new blood vessels from a pre-existing vascular bed, termed "angiogenesis," is of critical importance for the growth and development of the animal since it is required for the growth of the skeleton during endochondral ossification, development and cycling of the corpus luteum and uterus, and for the repair of tissues during wound healing. "Vasculogenesis," the de novo formation of blood vessels is also important for the proper function and development of the vascular system in the embryo. New blood vessel formation is a prominent feature and permissive factor in the relentless progression of many human diseases, one of the most important examples of which is neoplasia. It is for this reason that angiogenesis is considered to be one of the hallmarks of cancer. The development of new classes of drugs that inhibit the growth and proper functioning of new blood vessels in vivo is likely to provide significant therapeutic benefit in the treatment of cancer, as well as other conditions where angiogenesis is a strong driver to the disease process. During the preclinical safety testing of these drugs, it is becoming increasingly clear that their in vivo efficacy is reflected in the profile of "expected toxicity" (resulting from pharmacology) observed in laboratory animals, so much so, that this profile of "desired" toxicity may act as a signature for their anti-angiogenic effect. In this article we review the major mechanisms controlling angiogenesis and its role during endochondral ossification. We also review the effects of perturbation of endochondral ossification through four mechanisms-inhibition of vascular endothelial growth factor (VEGF), pp60 c-Src kinase and matrix metalloproteinases as well as disruption of the blood supply with vascular targeting agents. Inhibition through each of these mechanisms appears to have broadly similar effects on the epiphyseal growth plate characterised by thickening due to the retention of hypertrophic chondrocytes resulting from the inhibition of angiogenesis. In contrast, in the metaphysis there are differing effects reflecting the specific role of these targets at this site.     

Signaling pathways induced by vascular endothelial growth factor (review)

Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. Angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, while vasculogenesis is the development of new blood vessels from the differentiation of endothelial precursors (angioblasts) in situ. Vascular endothelial growth factor (VEGF) family members are major mediators of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to promote endothelial cell viability, mitogenesis, chemotaxis, and vascular permeability. It mediates its activity mainly via two tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR), although other receptors, such as neuropilin-1 and -2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4) binds VEGF-C and VEGF-D and is more important in the development of lymphatic vessels. While the functional effects of VEGF on endothelial cells has been well studied, not as much is known about VEGF signaling. This review summarizes the different pathways known to be involved in VEGF signal transduction and the biological responses triggered by the VEGF signaling cascade.     

Neovascular Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a condition that may cause blindness. The prevalence of the disease in the Western world is estimated at 1-2% of the population. Over the past decade, treatment of neovascular AMD has been shifting from destruction of newly formed blood vessels towards inhibitors that silence the vascular endothelial growth factor (VEGF) pathway. Such agents are often first-in-class biopharmaceuticals that benefit from the fact that they can be locally administered in an immune-privileged environment with slow clearance. These new VEGF pathway inhibitors have improved therapeutic effects over conventional treatment and have promoted the identification of novel targets for inhibition of AMD angiogenesis. This review describes the rationale behind the shift from conventional to current treatment options and discusses investigational, most notably biopharmaceutical, drugs that are in clinical trials. It also provides possible points for improvement of these treatments, specifically regarding their delivery.     

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti-VEGF antibodies indicated that other angiogenic factors may be involved. VEGF-D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor-2 (VEGFR-2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor-binding domain of VEGF-D and the use of these antibodies to localize VEGF-D in malignant melanoma. VEGF-D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF-D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF-D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR-2 and VEGFR-3. As VEGF receptors can be up-regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF-D in smooth muscle of the blood vessels suggest that VEGF-D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells.     

Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.