Arja Kaipainen

A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.

PPARγ ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis

Several drugs approved for a variety of indications have been shown to exhibit antiangiogenic effects. Our study focuses on the PPARgamma ligand rosiglitazone, a compound widely used in the treatment of type 2 diabetes. We demonstrate, for the first time to our knowledge, that PPARgamma is highly expressed in tumor endothelium and is activated by rosiglitazone in cultured endothelial cells. Furthermore, we show that rosiglitazone suppresses primary tumor growth and metastasis by both direct and indirect antiangiogenic effects. Rosiglitazone inhibits bovine capillary endothelial cell but not tumor cell proliferation at low doses in vitro and decreases VEGF production by tumor cells. In our in vivo studies, rosiglitazone suppresses angiogenesis in the chick chorioallantoic membrane, in the avascular cornea, and in a variety of primary tumors. These results suggest that PPARgamma ligands may be useful in treating angiogenic diseases such as cancer by inhibiting angiogenesis.

Semaphorin 3F, a chemorepulsant for endothelial cells, induces a poorly vascularized, encapsulated, nonmetastatic tumor phenotype

Melanoma is the most lethal skin cancer. Most deaths from melanoma result from metastases. Semaphorins have been shown to inhibit neuronal and endothelial cell migration, but the effects of semaphorins on tumor metastasis have not been documented. We found that semaphorin 3F (SEMA3F) was markedly downregulated in highly metastatic human cell lines in vitro and in vivo, which suggested that it may be a metastasis inhibitor. Metastatic human melanoma cells were transfected with SEMA3F and implanted into mice; the resultant tumors did not metastasize. Rather, the primary tumors resembled benign nevi characterized by large areas of apoptosis, diminished vascularity, inhibition of hyperplasia in overlying epidermal cells, and encapsulated tumor borders delineated by thick layers of fibroblasts and collagen matrix. This phenotype is in stark contrast to highly invasive, vascular mock-transfected tumors. In vitro, tumor cells expressing SEMA3F had a diminished capacity to adhere and migrate on fibronectin. Consistent with semaphorin-mediated chemorepulsion of neurons, tumor cells expressing SEMA3F were chemorepulsive for vascular and lymphatic endothelial cells expressing neuropilin-2 (NRP2), a novel mechanism for a tumor angiogenesis inhibitor. The repulsive activity was abrogated by NRP2 RNA interference. Together these results indicate that SEMA3F is a potent metastasis inhibitor that targets both tumor and stromal cells and raise the possibility of SEMA3F having therapeutic potential.

Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

PPARα agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition

Angiogenesis and inflammation are central processes through which the tumor microenvironment influences tumor growth. We have demonstrated recently that peroxisome proliferator-activated receptor (PPAR)α deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of thrombospondin (TSP)-1 and prevents tumor growth. Hence, we speculated that pharmacologic activation of PPARα would promote tumor growth. Surprisingly, the PPARα agonist fenofibrate potently suppressed primary tumor growth in mice. This effect was not mediated by cancer-cell-autonomous antiproliferative mechanisms but by the inhibition of angiogenesis and inflammation in the host tissue. Although PPARα-deficient tumors were still susceptible to fenofibrate, absence of PPARα in the host animal abrogated the potent antitumor effect of fenofibrate. In addition, fenofibrate suppressed endothelial cell proliferation and VEGF production, increased TSP-1 and endostatin, and inhibited corneal neovascularization. Thus, both genetic abrogation of PPARα as well as its activation by ligands cause tumor suppression via overlapping antiangiogenic pathways. These findings reveal the potential utility of the well tolerated PPARα agonists beyond their use as lipid-lowering drugs in anticancer therapy. Our results provide a mechanistic rationale for evaluating the clinical benefits of PPARα agonists in cancer treatment, alone and in combination with other therapies.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Vascular endothelial growth factors VEGF‐B and VEGF‐C

Vascular endothelial growth factor, which was idenheart cDNA library, respectively, by using a serendipitously found partial mouse cDNA clone as a probe tified almost 10 years ago, has so far been considered as the only growth factor relatively specific for endothelial (20,24). Independently, another group found the same gene when attempting to identify candidate genes for cells. VEGF is an important regulator of endothelial cell proliferation, migration, and permeability during multiple endocrine neoplasia type 1 (MEN1). The product of this alternatively spliced gene was designated as embryonic vasculogenesis and in physiological and pathological angiogenesis [reviewed in (1–3)]. The pivVRF (21). The two currently known isoforms of VEGF-B are otal role of VEGF in embryogenesis is emphasized by the unprecedented result that the inactivation of even a generated by alternative splicing of mRNA from the VEGF-B gene, spanning about 4 kb of DNA. The human single VEGF allele results in embryonic lethality (4,5). VEGF acts through its two known high-affinity recepand murine VEGF-B genes are composed of 7 exons, and their exon–intron organization resembles that of tors Flt1, vascular endothelial growth factor receptor 1 (VEGFR-1) and KDR/Flk1, or VEGFR-2 (6–10). A VEGF and PlGF genes (21,24,25). The mature VEGFB proteins (devoid of signal sequence) have 167 (VEGFthird receptor tyrosine kinase homologous with VEGFR-1 and VEGFR-2, designated Flt4, was cloned B167) and 186 (VEGF-B186) amino acid residues, respectively. VEGF-B186 is generated by using an alternative as an orphan receptor by two research groups and was shown not to bind VEGF (11–14). Additional VEGF splice acceptor site in exon 6, resulting in an insertion of 101 bp between nucleotides 410 and 411 in the coding receptors of unknown nature also exist on endothelial and tumor cells (15,16). sequence of VEGF-B167. This insertion introduces a frame shift and a stop codon at the position correspondThe second member of the VEGF family of growth factors, placenta growth factor (PlGF), is 53% identical ing to nucleotides 521–523 of the coding region of VEGF-B167 cDNA (Fig. 1). Thus, the two VEGF-B isowith VEGF within its platelet-derived growth factorlike region and binds only VEGFR-1 (17–19). Both forms have an identical NH2-terminal domain of 115 aa and different COOH-terminal domains. Although VEGF and PlGF are dimeric glycoproteins related in structure to the platelet-derived growth factors A and the C-terminus of VEGF-B167 is highly basic, that of VEGF-B186 is rich in alanine, proline, serine, and threoB (PDGF-A and PDGF-B). This relation is based on the presence of several conserved amino acid residues nine amino acid residues and has no significant similarity with amino acid sequences of known proteins including 8 equally spaced cysteines. Compared with VEGF, the mitogenic or permeability-enhancing activi(21,24). Unlike other growth factors of the VEGF-family, both isoforms of human and mouse VEGF-B lack ties of PlGF are weak; however, PlGF is able to potentiate the action of VEGF in vivo and in vitro (19). PlGF– the consensus sequence for N-linked glycosylation (NXT/S); instead, VEGF186 is O-glycosylated (24). VEGF heterodimers occur in vivo and have intermediate potency in mitogenic stimulation of endothelial VEGF-B167 remains cell associated with secretion, but it is released into the culture medium with treatcells (35). Two novel endothelial cell-specific growth factors, ment of the producing cells with heparin or high salt. The cell (or matrix) association of VEGF-B167 likely ocstructurally related to VEGF and PlGF, were recently discovered. These factors, designated as vascular endocurs via its basic region, as observed for the highly basic splice variants of VEGF. This notion is supported thelial growth factor B (VEGF-B) or VEGF-related factor (20,21) and vascular endothelial growth factor C by the fact that VEGF-B186 , lacking the highly basic region, is freely secreted from cells and is not bound to (VEGF-C) or VEGF-related protein (22,23) expand the known VEGF family and demonstrate the complexity cell-surface or pericellular heparan sulfate proteoglycans (20,24). of regulation of endothelial functions. This review summarizes the initial studies on VEGF-B and VEGF-C. The apparent molecular masses of the secreted VEGF-B167 and VEGF-B186 polypeptides are 21 kDa VEGF-B/VRF

Tensile forces stimulate vascular remodeling and epidermal cell proliferation in living skin.

Objectives:To quantify tissue remodeling induced by static and cyclical application of tensional forces in a living perfused tissue. Background:Cells are able to respond to mechanical cues from the environment and can switch between proliferation and quiescence. However, the effects of different regimens of tension on living, perfused skin have not been characterized. Methods:The ears of living rats were mechanically loaded by applying tensile forces (0.5 Newtons) either statically or cyclically and then analyzing tissue responses using in vivo microscopy, immunohistochemistry, and corrosion casting. Results:Quantitative immunohistochemistry showed that in the static group (4-day continuous tension) there was up to 4-fold increase in cellular proliferation in the epidermis after 4 days and a 2.8-fold increase in the vascularity in the dermis that peaked after 2 days. Comparable effects could be achieved in just 8 hours using a cyclic loading protocol. We also modeled the resultant stress produced in the ear using a linear finite element model and demonstrated a correlation between the level of applied stress and both epidermal cell proliferation and blood vessel density. Conclusions:Mechanical forces stimulate cell proliferation and vascular remodeling in living skin. As cell growth and vascular supply are critical to wound healing and tissue expansion, devices applying controlled mechanical loads to tissues may be a powerful therapy to treat tissue defects.

Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed.

Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation

Interstitial fluid movement is intrinsically linked to lymphatic drainage. However, their relationship is poorly understood, and associated pathologies are mostly untreatable. In this work we test the hypothesis that bulk tissue fluid movement can be evaluated in situ and described by a linear biphasic theory which integrates the regulatory function of the lymphatics with the mechanical stresses of the tissue. To accomplish this, we develop a novel experimental and theoretical model using the skin of the mouse tail. We then use the model to demonstrate how interstitial-lymphatic fluid movement depends on a balance between the elasticity, hydraulic conductivity, and lymphatic conductance as well as to demonstrate how chronic swelling (edema) alters the equipoise between tissue fluid balance parameters. Specifically, tissue fluid equilibrium is perturbed with a continuous interstitial infusion of saline into the tip of the tail. The resulting gradients in tissue stress are measured in terms of interstitial fluid pressure using a servo-null system. These measurements are then fit to the theory to provide in vivo estimates of the tissue hydraulic conductivity, elastic modulus, and overall resistance to lymphatic drainage. Additional experiments are performed on edematous tails to show that although chronic swelling causes an increase in the hydraulic conductivity, its greatly increased distensibility (due to matrix remodeling) dampens the driving forces for fluid movement and leads to fluid stagnation. This model is useful for examining potential treatments for edema and lymphatic disorders as well as substances which may alter tissue fluid balance and/or lymphatic drainage.