Cancer Angiogenesis and Its Master Regulator Perlecan

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.

We have recently demonstrated that granulocyte colony-stimulating factor (G-CSF) stimulated the production of epithelial-cell-derived-neutrophil attractant-78 (ENA-78) by neutrophils and that ENA-78 might promote the accumulation of neutrophils that had migrated from the intravascular space into inflammatory tissues. In this study, we examined whether other chemokines could be secreted by neutrophils that had accumulated after migrating from the intravascular space into the inflammatory tissues. We demonstrated that adhesion to laminin contained in the basement membrane and Matrigel, which is an artificial basement membrane model, induced macrophage inflammatory protein-1 beta (MIP-1 beta) in neutrophils and that MIP-1 beta secreted by neutrophils induced the chemotaxis of dendritic cells. These findings suggest that neutrophils transmigrating through the basement membrane are stimulated to secrete MIP-1 beta by the basement membrane, inducing the transmigration of dendritic cells from the intravascular space into inflammatory tissues. We propose that neutrophils intervene between innate immune response and specific immune response by secreting MIP-1 beta during the transmigration through the basement membrane.

To obtain data on the relative incidence of changes in the basement membrane of small peripheral blood vessels in diabetic and nondiabetic patients, a “blind” study of skin biopsies was undertaken. Parallel to a thorough clinical and laboratory work-up, punch biopsies of skin were obtained under local block anesthesia. A number of morphological features were analyzed by light and electron microscopy and graded on a semiquantitative scale, and the basement membrane was measured with calipers. The tabulated data were then correlated with clinical information. The following conclusions may be drawn: (a) “Capillaries” of the dorsal skin of the finger revealed a more complex structure than that seen in capillaries of muscle or loose connective tissue. This increased complexity was mainly due to incomplete reduplication of the endothelial cell wall by long abluminal cytoplasmic extensions. (b) The fine structure of the basement membrane as well as its thickness revealed very large variations, having one of the following appearances: (1) Homogeneous or finely fibrillar, and thin (2) Homogeneous or finely fibrillar, and thick (3) Finely fibrillar and lamellated (4) Containing inclusions. (c) Careful, unbiased evaluation of “capillaries” of control patients without family or own history or chemical evidence of diabetes and who can reasonably be classified as normal, and of diabetic patients, leads to results permitting no distinction between the two groups. No differences could be seen regarding the number of endothelial and/or pericytic cells per “capillary,” state of vascular contraction or dilatation, cytologic detail, or type or width of the basement membrane between normal controls and diabetic patients. (d) The reasons for the variations in basement membrane thickness and appearance and in cellular structure are not known, but diabetes can probably be excluded as a specific etiologic factor. (e) The results of this investigation again underline the importance of “blind” studies with adequate controls, especially where subjective evaluation cannot be excluded. (f) It may be advisable to conduct similar blind studies on other capillary territories to assess in an unequivocal manner the relationship between the appearance of the endothelium and the basement membrane and specific diseases.

Blood pressure as a potential biomarker of the efficacy angiogenesis inhibitor

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.

The maintenance of tissues of virtually all organs depends on a sufficient blood supply. During embryogenesis, primitive blood vessels are formed de novo by the aggregation of angioblasts, a process that is termed vasculogenesis. In postnatal life, the development of new blood vessels is restricted to the female reproductive tract (during the ovulatory cycle) and to sites of wound healing, and occurs through a process called angiogenesis, i.e. the sprouting of new vessels from the preexisting vasculature. However, neovascularization can also occur under pathological conditions, e.g. tumor cells can "switch on" angiogenesis. New blood vessels bring in nutrients and proteins, so the tumor mass can expand. In fact, neovascularization appears to be one of the crucial steps in the transition of a tumor from a small cluster of malignant cells to a visible macroscopic tumor capable of spreading to other organs via the vasculature throughout the body. The association of tumor growth with the development of a vascular network was recognized nearly a century ago. Using a leukemia model, chronic myelogenous leukemia (CML), we were able to provide evidence for the existence of a hemangioblastic progenitor cell in the bone marrow of adult humans. Using the pathognomonic BCR-ABL-fusion gene as a genetic marker present in virtually all bone marrow derived cells of patients with CML, we were able to show that endothelial cells belong to the malignant cell clone, since they also contain the BCR-ABL-fusion gene. Our data suggest that CML arises from a hemangioblastic progenitor cell, the progeny of which are malignant blood cells and genotypically clonal endothelial cells. Thus, we provide substantial evidence that indeed a hemangioblast exists in the bone marrow of human adults. In addition, our data imply that normal as well as genotypically malignant bone-marrow-derived endothelial cells can contribute to maintenance angiogenesis in the vascular endothelium, a condition that is consistent with postnatal vasculogenesis. These findings were recently confirmed by other groups and should help in elucidating the pathophysiology of malignant and nonmalignant disorders. The integration of bone-marrow-derived endothelial cells into the vascular endothelium has implications for the development of vascular targeting strategies (e.g., gene therapy) for vascular diseases, inflammatory disorders, and cancer. The characterization of the hemangioblast at a clonal level as well as the translation of these findings into a clinically applicable concept for the delivery of therapeutic genes to malignant tumors is currently in progress in our laboratory.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

A poorly differentiated medullary carcinoma of human stomach, designated HY–1, was successfully transplanted to nude mice by either the subcutaneous or intramuscular route for five generations. The transplanted tumor showed spontaneous lung metastases in nearly 100% of KSN and Balb/c female nude mice. There were over 20 visible lung metastatic nodules in KSN and Balb/c nude mice bearing tumors for over SO days. Immunostaining of type IV collagen and electron microscopy revealed that tumor cells were often in direct contact with basement membrane (BM) of tumor blood vessels in the primary tumor tissue. At the site of contact between tumor cells and vascular BM, focal disappearance of the BM, disruption of endothelial cells and entry of tumor cell clusters into vascular lumen were observed. Immunostaining of 72 kDa gelatinase/type IV collagenase demonstrated that tumor cells expressed this enzyme in their cytoplasm. These results suggest that spontaneous metastasis of this tumor may be partly due to a marked tendency to vascular invasion involving the following sequential events: tumor cell contact with vascular BM, BM degradation possibly by 72 kDa gelatinases and endothelial disruption. This model could be a useful tool for understanding the mechanism of hematogenous metastasis of human gastric cancer.

Endothelial cell invasion is an essential event during angiogenesis (the formation of new blood vessels). This process involves the degradation of the extracellular matrix, the basement membrane, and interstitial stroma, and is governed by the activation of matrix metalloproteinases. However, the contribution of matrix metalloproteinases in angiogenesis is much more complicated. Tumor growth above a certain size is dependent on new vessels. A number of studies have demonstrated that treating tumors with matrix metalloproteinase inhibitors results in tumor reduction and a decrease in tumor angiogenesis. Matrix metalloproteinases as sole matrix eaters or degraders is a matter of the past. Not only tumor cells but more importantly bystander cells such as stromal cells produce matrix metalloproteinases. Matrix metalloproteinases therefore are also part of the pathologic microenvironment in different diseases. This enzymatic microenvironment dictates the endothelial cell fate, the angiogenic switch, and finally angiogenesis. During recent years, the role of matrix metalloproteinases has expanded, and their function as modulators of biologically active signaling molecules has drawn much attention. Depending on their substrate (growth factors or their receptors, extracellular matrix components, and angiogenic factors), matrix metalloproteinase activation results in the generation of proangiogenic or antiangiogenic factors. These data challenge the old concept that matrix metalloproteinases are simply proangiogenic. The knowledge of the local enzymatic profile and what, where, and how matrix metalloproteinases are involved in angiogenesis of tumors or other diseases will help design future therapeutic strategies better reflecting the complexity of the underlying biologic process of angiogenesis.

The morphological basis of proestrus endometrial bleeding in canines

Endothelium-Microenvironment Interactions in the Developing Embryo and in the Adult

Matrix metalloproteinases (MMPs), also termed matrixins, are known to affect atherosclerosis in many diverse ways.1–3 Apart from a role in the progression of atherosclerotic lesions, MMPs have also been involved in plaque destabilization and rupture and in the development of aneurysms. Despite intense research efforts during the last decade, it has been challenging to formulate a unifying model on the specific role of MMPs in atherosclerosis. In fact, mouse genetic studies have yielded controversial insights, highly dependent on genetic background, dietary regimen, and arterial site of analysis, whereas human genetic association studies do not prove causality of MMPs in atherosclerosis. Moreover, the lack of specific inhibitors precluded pharmacological dissection of the role of MMPs in vivo. There is thus a need for alternative strategies to decipher the MMP puzzle in atherosclerosis. In this issue of Circulation , Liang et al4 used a novel approach: They generated transgenic rabbits to study the role of MMP-12, also known as macrophage elastase, in atherosclerosis. Their results indicate that MMP-12 causes media destruction and pseudoaneurysm formation and, more surprisingly, accelerates plaque growth in an animal model that more closely resembles atherosclerosis in humans. Article p 1993 MMPs belong to one of the most ancient families of enzymes that arose in evolution soon after the first signs of life to regulate, in general, interactions of cells with the extracellular matrix (ECM). To cope with this formidable challenge, more than 65 structurally related MMPs developed in bacteria, plants, nematodes, fruit flies, sea urchins, and vertebrates. One of their key activities is breaking down ECM substrates in the basement membrane and interstitial matrix.1,2 As such, MMPs facilitate migration of macrophages and smooth muscle cells (SMCs) in atherosclerotic plaques by degrading ECM barriers, exposing novel ECM adhesion sites, and relieving the binding between basement membrane …

Perlecan is a major heparan sulfate proteoglycan (HSPG) of basement membranes (BMs) and connective tissues. The core protein of perlecan is divided into five domains based on sequence homology to other known proteins. Commonly, the N-terminal domain I of mammalian perlecan is substituted with three HS chains that can bind a number of matrix molecules, cytokines, and growth factors. Perlecan is essential for metazoan life, as shown by genetic manipulations of nematodes, insects, and mice. There are also known human mutations that can be lethal. In vertebrates, new functions of perlecan emerged with the acquisition of a closed vascular system and skeletal connective tissues. Many of perlecan's functions may be related to the binding and presentation of growth factors to high-affinity tyrosine kinase (TK) receptors. Data are accumulating, as discussed here, that similar growth factor-mediated processes may have unwanted promoting effects on tumor cell proliferation and tumor angiogenesis. Understanding of these attributes at the molecular level may offer opportunities for therapeutic intervention.

In the most common primary brain tumors, malignant glioma cells invade the extracellular matrix (ECM) and proliferate rapidly in the cerebral tissue, which is mainly composed of hyaluronan (HA) along with the elastin present in the basement membrane of blood vessels. To determine the role of ECM components in the invasive capacity of glioma cell lines, we developed a 3-D cell-culture system, based on a hydrogel in which HA can be coreticulated with kappa-elastin (HA-kappaE). Using this system, the invasiveness of cells from four glioma cell lines was dramatically increased by the presence of kappaE and a related, specific peptide (VGVAPG)(3). In addition, MMP-2 secretion increased and MMP-12 synthesis occurred. Extracellular injections of kappaE or (VGVAPG)(3) provoked a pronounced and dose-dependent increase in [Ca(2+)](i). kappaE significantly enhanced the expression of the genes encoding elastin-receptor and tropoelastin. We propose the existence of a positive feedback loop in which degradation of elastin generates fragments that stimulate synthesis of tropoelastin followed by further degradation as well as migration and proliferation of the very cells responsible for degradation. All steps in this ECM-based loop could be blocked by the addition of either of the EBP antagonists, lactose, and V-14 peptide, suggesting that the loop itself should be considered as a new therapeutic target.

Electron microscopic development of neuroglia in epiretinal portion of post-natal rabbits