Introduction and application

The tremendous vascular surface area represented by the pulmonary endothelium aids in essential blood/gas exchange but also effectively regulates circulating levels of vasoactive agents. For example, protease activities on the endothelial surface can dictate total blood levels of critical vasoregulatory peptides such as angiotensin converting enzyme (ACE). The continuous capillaries of the lung comprise the majority of this surface area and maintain a highly selective permeability barrier that, when compromised, can lead to oedema and pulmonary pathology.

The microvessels of the lung undoubtedly develop some of their physiological activities from interactions with other pulmonary cells. Information exchange with epithelial type I and type II cells occurs at the level of secreted and extracellular matrix proteins to dictate proper airway architecture and vascular function. It is the understanding at this cellular level that has driven researchers to take the simplistic approach of isolating cell types and culturing them in vitro. But how much information do isolated and repeatedly cultured cells ‘remember’ about how to mimic their functions in situ? If this question is asked frequently by cell and molecular biologists who desire the optimal in vitro model of a vascular bed, our understanding of whole-organ physiology will consequently benefit.

The application of in vitro models of the pulmonary microvasculature must be tempered with defining the cell type as well as the desired physiological measurement. For example, the use of ACE as a reliable marker for pulmonary endothelium was aided by the knowledge that angiotensin could be cleaved when passed through an isolated lung preparation.

Introduction

For many years the endothelial cell was regarded in medicine and physiology as ‘an inert sac whose function was simply to separate the blood from the tissues’. We now know that the endothelium is an extremely active tissue that plays an intimate role in many physiological processes. Dysfunction of the endothelial cell is a key step in the development of many pathologies including the two major killers of Western man: atherosclerosis (a disease of the larger vessels) and cancer (tumour angiogenesis). As the skill of biologists in the characterisation of the endothelium in vivo and in vitro has increased it has become apparent that there exists extensive endothelial heterogeneity. Thus, the tenet that an endothelial cell is an endothelial cell is an endothelial cell is no longer valid. A corollary of this heterogeneity is that it is imperative to work with an appropriate end othelial isolate when studying the role of endothelium in a given pathology. From this has sprung the increasing interest in the isolation and culture of endothelium from diverse organs.

A remarkable property of endothelium is its quiescence. Thus, in the adult the average endothelial cell divides (approximately) twice in a lifetime. Contrast this with epithelia that divide continually throughout adult life. Nevertheless, when required, endothelial proliferation can be very rapid. Consider a healing wound: endothelial proliferation (as a component of angiogenesis) is switched on, then off, as required and is under exquisite control. In the past I have likened this angiogenesis to the blood clotting cascade. The existing vasculature is a coiled spring poised for angiogenesis should a physiological need arise.

Introduction

Brain microvascular endothelial cells have successfully been maintained in culture for some time by many groups, but it has proved to be very difficult to establish a true in vitro model of the blood–brain barrier (BBB). Although a few promising protocols have been published fairly recently, reproducibility appears to be problematic as judged by the scarcity of studies in which these protocols have been adopted.

The present chapter does not aim at providing an overview on brain endothelial cells in culture. For this the reader is referred to reviews by Rubin (1991), Joó (1992) and Laterra & Goldstein (1992, 1993). Nor is it the intention to present a new protocol for the isolation and growth of these cells in culture. One protocol is given at the end of the chapter, otherwise the reader will be referred to the literature. Instead the focus will be on specific problems associated with the pursuit of establishing an in vitro model of the BBB, mainly on aspects such as differentiation of endothelial cells in culture and on evaluation and characterisation of such culture systems. It is meant to be a guide for those who plan to grow brain microvascular endothelial cells in culture.

Brain versus peripheral endothelial cells

Paul Ehrlich observed in 1885 that dye injected into the vasculature of animals readily penetrated all tissues except the brain. About 80 years later it was shown in similar tracer experiments that the structural basis for this ‘blood–brain barrier’ is the endothelial cells which constitute the microvascular walls in the brain.

Introduction

That endothelial cells derived from different vascular organs, and from within different vascular beds within those organs, display morphological, biochemical and antigenic heterogeneity is now beyond dispute (Kumar et al, 1987; Zetter, 1988; Kuzu et al., 1992; Hewett & Murray, 1993a). This fact has highlighted the need for methods to isolate and maintain in culture endothelial cells from a variety of species and tissues. Unquestionably the richest sources of microvascular endothelial cells have been tissues such as adipose tissues and brain which, by virtue of the special physical characteristics of their parenchyma, allow the ready separation of microvessels from the bulk of the tissue. The difference in buoyant densities of adipocytes and the stromal component of adipose tissues was first exploited by Wagner & Matthews (1975) for the isolation of microvessel endothelium from the rat epididymal fat pad. Similarly, high microvessel density, ease of separation, and the resistance of the parenchyma to growth in routine culture, have made brain a relatively accessible source of microvessel endothelium (Kumar et al., 1987; Hewett & Murray, 1993a). Nevertheless the isolation of endothelial cells from more complex tissues with a wide variety of cell types, many of which readily adapt to cell culture in competition with the endothelial cell of interest, has until recently remained a significant technical problem. The development of new techniques producing relatively large numbers of pure endothelial cells has in addition led to the realisation that endothelial cells are less fastidious in terms of growth requirements than previously thought, and microvessel cells from a wide variety of tissues can now be routinely cultured.

Introduction

The pioneering work of Lewis in the 1920s provided the groundwork for the eventual establishment of both large blood vessel and microvessel derived endothelial cell cultures (Lewis, 1922). The earliest report of methods for the isolation of microvessel derived endothelial cells, and in particular endothelial cells derived from microvascularised fat, was a publication by Wagner and colleagues in 1972. The choice of fat as a source of tissue was based on its ready availability, its high density of microvascular endothelial cells and previously established methods for the digestion of adipose tissue using proteolytic enzyme solutions containing crude clostridial collagenase. Thus the choice of fat tissue as a source of endothelium was not based on specific properties to be studied. In fact Wagner's report provided initial evidence that not only microvascular endothelium but endothelium in general exhibited specific metabolic activities. The tenet that endothelium existed as a metabolically inert ‘Cellophane’ lining was initially questioned by Wagner's evaluation of isolated fat-derived microvascular endothelial cells.

Since these early studies using adipose tissue derived endothelium, investigators have begun to evaluate microvascular endothelial cells from essentially every vascularised tissue in the body. Continued interest in adipose tissue endothelial cells persists for the following reasons: (1) studies of obesity and general nutrition, (2) studies of the augmentation of wound healing by the presence of adipose tissue and (3) the use of adipose tissue derived endothelium for cell transplantation. Obesity and nutrition studies have utilised adipose-derived endothelium to evaluate their role in anabolic and catabolic activity and in the regulation of nutrient and waste product transport between blood and adipocytes.

Introduction

Bone marrow is a highly compartmentalised structure in which haemopoiesis occurs within distinct anatomical niches. Endothelial cells of the bone marrow microvasculature are thought to play a central role in regulating cellular traffic between the intravascular and extravascular (haemopoietic) compartments of the bone marrow (Tavassoli, 1979). The volume of this cellular traffic is highly variable, according to demands such as blood loss or infection. Normal haemopoiesis requires intimate contact between marrow stromal cells and haemopoietic progenitor cells (HPCs). Maturing HPCs detach from the stroma, traverse the sinusoidal endothelium and enter the circulation.

Circulating HPCs are capable of selective ‘homing’ to the bone marrow (Tavassoli & Hardy, 1990). This process is essential to the clinical practice of bone marrow transplantation, in which intravenously transplanted progenitors return to the extravascular compartment of the bone marrow and restore multilineage haemopoiesis. Homing is also thought to occur under normal conditions of haemopoiesis. The homing process is thought to occur in two stages. In the first step, circulating HPCs interact with the luminal surface of sinus-lining endothelium. The cells then migrate through the endothelial layer and enter the extravascular compartment, where they interact with stromal cells, extracellular matrix components, and growth factors.

Interactions between various stromal components of the bone marrow, and HPCs are well documented, and the role of these interactions in progenitor cell homing has been the subject of a large number of studies. By contrast, little is known about the role of microvascular endothelium of the bone marrow in the homing process.

Introduction

Contrary to popular belief the first attempts to culture endothelial cells in vitro pre-date considerably the work of Jaffé et al. (1973) or Gimbrone and others in the early 1970s. Some 50 years earlier in 1922, Warren Lewis reported the growth of endothelial cells isolated from chick liver sinusoids, and in 1925 Maximov demonstrated growth of vessels from rabbit pia. However, such cultures were essentially organ cultures which relied on high plasma concentrations to support the limited ‘endothelial’ like cell outgrowth from the explanted tissue. The work of Jaffé, Gimbrone and colleagues deserves mention for their pioneering work in isolating and maintaining endothelial cells in long-term culture as a single cell type, free of fibroblastic, and other non-endothelial, cell contamination.

To examine the role of particular microvascular beds in certain disease states, the long-term culture of endothelial cells derived from human microvascular beds remains a primary goal for many cell biologists. The realisation of this goal was aided immensely by the seminal articles of Judah Folkman and colleagues in the late 1970s (Folkman et al. 1979) which defined the fastidious growth conditions required by certain human microvascular endothelial cells in vitro. Unfortunately, the laborious isolation methods employed by Folkman and Haudenschild have been less widely applicable. However, as this book highlights, there is no universal isolation and culture method which is applicable to all microvascular endothelial cell sources.

Theoretically, if a vessel can be cannulated, endothelial cells can be isolated by collagenase perfusion in a manner similar to that proposed by Jaffé for umbilical veins (Jaffe et al., 1973).

Introduction

The vasculature of the female reproductive tract is intimately involved in the processes of ovulation, menstruation, implantation, placentation and fetal development. These tissue beds have the unique property of undergoing benign angiogenesis, a process otherwise restricted to tissue repair. This chapter will describe the isolation and culture of the endothelium of the corpus luteum and of human first trimester (early pregnancy) decidua. It will also briefly cover the endothelium of other female reproductive tissues. No description will be given of the isolation and culture of human umbilical vein endothelium (HUVEC) since this has been described in Chapter 3.

The ovary

Mammalian ovarian follicles possess a well-developed capillary bed that delivers nutrients, growth factors and gonadotrophins required for the growth and maturation of the follicle and subsequent transformation into a corpus luteum. Ovarian follicles are thought to control the development of their own capillary bed, although there is no strong evidence to support this. Various regulators of angiogenesis have been identified in ovarian tissue, including epidermal growth factor (EGF), transforming growth factor α (TGFα), transforming growth factor β (TGFβ), basic fibroblast growth factor (bFGF), tumor necrosis factor α (TNFα), interleukin-1 (IL-1), inhibin and activin. The precise role played by each factor is not understood. Inhibition of ovarian angiogenesis could give rise to the development of new contraceptive agents.

During the menstrual and oestrous cycles, the ovarian follicle ruptures at ovulation and then develops into the corpus luteum which contains a well-developed microvasculature. Attempts have been made to isolate and culture endothelial cells from bovine and rabbit corpora lutea (Spanel-Borowski & Bosch, 1990; Bagavandoss & Wilks, 1991; Fenyves et al, 1993) and detailed methods are given in the following section.

The series Handbooks in Practical Animal Cell Biology was born out of a wish to provide scientists turning to cell biology, to answer specific biological questions, the same scope as those turning to molecular biology as a tool. Look on any molecular cell biology laboratory's bookshelf and you will find one or more multivolume works that provide excellent recipe books for molecular techniques. Practical cell biology normally has a much lower profile, usually with a few more theoretical books on the cell types being studied in that laboratory.

The aim of this series, then, is to provide a multivolume, recipe-book-style approach to cell biology. Individuals may wish to acquire one or more volumes for personal use. Laboratories are likely to find the whole series a valuable addition to the ‘in house’ technique base.

There is no doubt that a competent molecular cell biologist will need ‘green fingers’ and patience to succeed in the culture of many primary cell types. However, with our increasing knowledge of the molecular explanation for many complex biological processes, the need to study differentiated cell lineages in vitro becomes ever more fundamental to many research programmes. Many of the more tedious elements in cell biology have become less onerous due to the commercial availability of most reagents. Further, the element of ‘witchcraft’ involved in success in culturing particular primary cells has diminished as more individuals are successful. The chapters in each volume of the series are written by experts in the culture of each cell type. The specific aim of the series is to share that technical expertise with others. We, the editors and authors, wish you every success in achieving this.

Introduction

Local differentiation gives rise to remarkable endothelial heterogeneity in the vascular and lymphatic systems of different organs. Because of its strategic location, microvascular endothelium plays a central role in skin physiology, and its involvement in pathologies of the skin is becoming increasingly apparent. Thus, endothelial cells play a role in anti-thrombogenicity, blood vessel permeability, blood pressure control, metabolism of lipoproteins, tissue ageing, antigen presentation, and angiogenesis in wound healing. Dysfunctions are associated with a number of pathologies such as inflammation, immune disorders and hyperproliferation of vessels in, for example, psoriasis.

In vitro culture of endothelial cells from large vessels (aorta and umbilical vein) has provided a useful model for the study of endothelial cell metabolism; however, the validity of using endothelial cells isolated from the macrovasculature to study microvascular behaviour and function is questionable. Nearly 20 years ago, the group led by Karasek developed endothelial cell cultures derived from capillaries of rabbit skin (Davison & Karasek, 1978) and from the human newborn foreskin dermis. These established more representative in vitro models of the skin microvasculature (Davison et al, 1980). More recently, Davison et al. have also published a method for the isolation and long-term serial cultivation of endothelial cells derived from microvessels of the adult human dermis (Davison et al., 1983).

Cultured skin microvascular endothelial cells have a wide range of research applications including the study of cellular differentiation, cytokine expression, haemorrhagic disorders, hypersensitivity, inflammation, thrombogenesis and thrombosis, tumour metastasis, and wound healing. Because of a large reduction in toxicological tests involving laboratory animals, in vitro cell culture models are of increasing importance.

When I was first approached to write this book for the Studies in Biology series, I was delighted. As a student this series had a profound influence on me, and I was an avid reader of the books. They provided an easily affordable access to a very wide range of topics in biology, and I still use one or two books from my student days.

I was also very daunted at the prospect of writing such a short book to cover so vast a topic as microbiology. It is a subject that impinges on almost every aspect of human existence. To attempt to cover the whole of the subject would have been an impossible task. Consequently, some very difficult decisions have had to be made, and some very interesting material has had to be omitted. It is to be regretted that we could not expand upon topics such as the story of the near collapse of Winchester Cathedral as a result of fungal decomposition of the oak raft that supported the structure, following drainage of nearby farm-land.

My co-authors and I have confined ourselves to a consideration of the aspects of microbiology in which we have research experience: bacteriology, mycology and virology. We have also tried to concentrate on the fundamental problems in the subject. What constitutes microbes? How do they differ from higher organisms and from each other? How can microbes be controlled, visualised, enumerated and cultured?

Safe handling of microbes

The vast majority of microbial life on Earth is harmless to humans, and many microorganisms have beneficial effects. Since the dawn of civilisation, humans have harnessed microbial fermentations to make bread and alcoholic beverages, and to prolong the life of food. Today, technologists are exploiting microorganisms in the pharmaceutical industry, for food production, mineral extraction, the oil industry and in agriculture. There is hardly any aspect of modern life that is not touched by microbiology. However, a small minority of microbes do cause disease, and a minority of disease-causing microbes can cause fatal infections. Some, such as the human immunodeficiency virus, the cause of AIDS, may take several years to exert their lethal effect. Others such as Neisseria meningitidis, the cause of meningococcal meningitis, can kill within hours of the first symptoms of the disease.

When working with microbes, care must be taken to ensure that laboratory cultures do not escape to cause laboratory-acquired infections or to pollute the environment. Equally, it is important to ensure that laboratory cultures do not become contaminated with unwanted extraneous organisms from the environment. If care is not taken to avoid contamination of laboratory cultures, then the results of microbiological experiments are not reliably reproducible, and the data obtained would be unreliable. It is impossible to tell whether the observations made in such experiments are due to the properties of the desired organism, or arise from the activity of a contaminant.

Charged by its Royal Charter to promote biology and its understanding, the Institute of Biology recognises that it is not possible for any one text book to cover the entirety of a course. If evidence was needed, the success of the Studies in Biology series was a testimony to the need for specialist, up-to-date publications in education. The Institute is therefore pleased to collaborate with Cambridge University Press in producing a new title in the Studies in Biology series.

The new series is set to provide as great a boon to the new generation of students as the original did to their parents.

Suggestions and comments from readers will always be welcomed and should be addressed either to the Studies in Biology Editorial Board at Cambridge University Press or c/o The Books Committee at the Institute.