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Stem cell therapy is a progressive approach to a pervasive clinical problem; cardiovascular disease is the number 1 killer in the USA and other developed countries, and aspects of it are amenable to stem cell therapy. Many types of stem cells have been used in treating the heart during myocardial infarction, and here, we describe our approach of direct myocardial injection of bone marrow-derived mesenchymal stem cells into the infarct of rats. We will also briefly introduce the methods we have used to inject neonatal cardiomyocytes into the aorta as a first step in attempting to produce an external cardiac pump. Proper surgical technique and postoperative care are as important as adequate injection of the cells and will greatly improve the survival of the animal after surgery. By carefully following the methods presented in this chapter, the reader will be able to perform direct myocardial and vascular injection of stem cells into rats. [ABSTRACT FROM AUTHOR]

Proteomics technology allows a comprehensive and efficient analysis of the proteome and has become an indispensable tool in biomedical research. Since the late 80s, advances on mass spectrometry (MS) instrumentation and techniques have revolutionized the way proteins can be analyzed. Such analysis would only be possible with a proper sample preparation and separation ahead of the MS step. Different gel and nongel-based methods are available for protein separation. This chapter will focus on the use of two-dimensional gel electrophoresis (2-DE) in proteomics and its application to platelet research. 2-DE separates proteins according to their isoelectric point (pI) and size (molecular weight) and allows the detection of thousands of proteins at a time. Platelets are enucleated cells that play a critical function in the control of bleeding and wound healing. As platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. This chapter presents a protocol for an efficient sample preparation, protein separation by 2-DE, and protein digestion ahead of the MS analysis. The experimental approach, already successfully applied to the study of the platelet proteome, includes recommendations for an efficient platelet preparation for proteomics studies. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is the most common disease in Westernized countries in large part because of the rising prevalence of obesity and physical inactivity. In addition, diabetes mellitus is an important risk factor for both heart failure and ischemic heart disease. As insulin resistance is known as an important pathophysiological feature in the cardiac diseases, understanding the mechanisms responsible for altered metabolism and insulin signaling in the diabetic heart may help identify novel targets in these conditions. Phosphatidylinositol (PI)-3 kinase (PI3K) and Akt are key signaling molecules in insulin and insulin-like growth factor-1 (IGF-1), which induce multiple biological effects in the heart such as cell survival and hypertrophy. Here, we have shown several fundamental techniques to study the role of PI3K and Akt in heart diseases. [ABSTRACT FROM AUTHOR]

Advanced glycation end products are permanently modified protein derivatives formed in the presence of reducing sugars, such as glucose, fructose, hexose-phosphates, trioses, and triose-phosphates by non-enzymatic glycation and oxidation ("glycoxidation") reactions and further irreversible rearrangements. Numerous studies have revealed the pivotal role of protein glycoxidation in the pathogeneses of diabetes-related and age-related diseases. Protein glycoxidation is generally recognized both as a hallmark and as a promoter for progression of diabetes-related and age-related ailments, particularly in cardiovascular system such as increased vascular and myocardial stiffness, endothelial dysfunction, altered vascular injury responses, and atherosclerotic plaque formation. An appropriate surveillance on abnormal protein glycoxidation at an early stage of disease progression is of clinical and practical importance to handle diabetes-related and age-related cardiovascular complications especially those leading to ventricular dysfunction. [ABSTRACT FROM AUTHOR]

Reactive oxygen and nitrogen species are thought to contribute to pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, restenosis, heart failure, and diabetic vascular complications. Some of these reactive oxygen species also play an important role in vascular signaling. In this chapter, we describe various techniques that we have successfully employed to reliably measure superoxide and hydrogen peroxide. Because reactive oxygen species are capable of rapidly inactivating nitric oxide and because endothelial function characterized by nitric oxide bioavailability is an important indicator of vascular health, we have also included novel techniques capable of directly measuring nitric oxide radical from vascular cells and tissues. [ABSTRACT FROM AUTHOR]

Migration, proliferation, and collagen synthesis by vascular smooth muscle cells are thought to be key events involved in the pathogenesis of cardiovascular disease. Following endothelial injury, smooth muscle cells (SMCs) in the intima of the blood vessels assume a synthetic, pro-mitogenic phenotype resulting in their migration, proliferation, and deposition of extracellular matrix within the neointimal tissue. This chapter describes a method of isolation of SMCs from rat aorta and in vitro assays to characterize these abnormal SMC functions. [ABSTRACT FROM AUTHOR]

Isolation of ventricular myocytes from all species of animals has revolutionized the field of cardiovascular research, allowing the assessment of true cardiac effects of drugs, treatments, and so on. With recent advances in physiology at the cellular level, direct assessment of isolated ventricular myocyte mechanics has become an increasingly powerful and important area of research. This technique provides important information on excitation-contraction coupling of the heart, either when investigating the effect of a drug on the heart or mechanical function under certain pathophysiological states. The goal of this chapter is to provide a detailed list of methods for isolation of ventricular myocytes from both rats and mice, as well as a list of certain "tricks" that have proven useful in enhancing the yield of viable cells from the whole heart. Mastering the isolation of myocytes will provide a much better framework for real-time beat-to-beat recording of myocyte contraction as well as intracellular Ca2+ transients. [ABSTRACT FROM AUTHOR]

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the local buildup of metabolic byproducts that occur in ischemic myocardium in vivo. Coverslip hypoxia (CSH) is a recently described method for studying hypoxia and ischemia derived from the byproducts and metabolites of contractile ventricular myocytes. Hence, this method is dependent on the purity and contractile activity of the isolated myocytes. We describe herein methods for isolating neonatal rat ventricular myocytes with these characteristics, as well as means for performing CSH, identifying viable and compromised myocytes after coverslipping, and tracking pH changes during CSH. [ABSTRACT FROM AUTHOR]

The pathogenesis of heart disease and the development of myocardial failure are highly dependent upon the cardiomyocyte—the basic contractile cell within the heart. Understanding and elucidating the complex networks that regulate cardiomyocyte function are central to the development of specific target-based therapeutic interventions. The relative recent advances in molecular genetics and generation and routine usage of transgenic and knockout mouse models have further necessitated that previously established cardiomyocyte methods be adapted for the isolation, culture, and study of primary adult murine cardiomyocytes, both freshly isolated and in culture. Such adaptation is based not only upon scalability of established techniques, as the mouse heart is an order of magnitude smaller than the rat heart, but also upon properties unique to the mouse. This chapter therefore describes current methods for the isolation, culture, and functional analysis of adult murine cardiomyocytes. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have emerged as potentially useful substrates for neovascularization and tissue repair and bioengineering. EPCs are a heterogeneous group of endothelial cell precursors originating in the hematopoietic compartment of the bone marrow. MSCs are a rare population of fibroblast-like cells derived from the bone marrow stroma, constituting approximately 0.001-0.01% of the nucleated cells in the marrow. Both cells types have been isolated from the bone marrow. In addition, EPC can be isolated from peripheral blood as well as the spleen, and MSC has also been isolated from peripheral adipose tissue. Several approaches have been used for the isolation of EPC and MSC, including density centrifugation and magnetic bead selection. Phenotypic characterization of both cell types is carried out using immunohistochemical detection and fluorescence-activated cell sorting analysis of cell-surface molecule expression. However, the lack of specific markers for each cell type renders their characterization difficult and ambiguous. In this chapter, we describe the methods that we use routinely for isolation, characterization, and genetic modification of EPC and MSC from human, rabbit, and mouse peripheral blood and bone marrow. [ABSTRACT FROM AUTHOR]

Endothelial cell (EC) migration plays an important role in embryonic vasculogenesis, angiogenesis, and wound healing. EC migration can be regulated by extracellular matrix (ECM) and hemodynamic forces through haptotaxis (induced by an ECM gradient) and mechanotaxis (induced by mechanical forces). Previously, the effects of haptotaxis or mechanotaxis alone on EC migration have been studied; however, the dual effect of haptotaxis and mechanotaxis on EC migration is not known. We developed a micropatterning technique to generate step changes of collagen surface density to monitor haptotactic EC migration. To investigate the crosstalk between haptotaxis and mechanotaxis on EC migration, we used an in vitro flow system to apply a well-defined fluid shear stress on ECs cultured on the micropatterned collagen. The study on the effects of haptotaxis and mechanotaxis on EC migration will provide a rational basis for promoting vascular wound healing, angiogenesis, and vascularization in engineered tissues. [ABSTRACT FROM AUTHOR]

This chapter describes a method that permits simultaneous measurement of leukocyte-endothelium interactions and endothelial nitric oxide (NO) levels in the microcirculation in vivo. The method is also useful to study the effect of NO replenishing therapy on adhesion of leukocytes to the vascular endothelium in acute and chronic inflammatory states of the cardiovascular system. This research approach requires the combination of two well-established physiology techniques, that is, intravital microscopy and real-time measurement of NO with microelectrodes. Intravital microscopy is considered the method of choice to monitor leukocyte-endothelial cell interactions in intact vascular beds of live animals. In vivo microscopy is currently used to study the endothelial cell phenotype of mice carrying mutations or deletion of targeted genes. Intravital microscopy is also used to study endothelial cell function in acute (e.g., ischemia-reperfusion injury) and chronic (e.g., hypercholesterolemia, hyperglycemia, and diabetes) inflammatory states of the cardiovascular system. NO sensors allow for continuous, amperometric quantification of NO levels in cells and organ tissues. Coupling of NO electrode technology with intravital microscopy has recently permitted to measure NO bioavailability in the normal and inflamed microcirculation. The method described here can be used to study in vivo how acute and chronic inflammatory states of the cardiovascular system alter endothelial function resulting in endothelial cell activation and damage. [ABSTRACT FROM AUTHOR]

Endothelial injury represents a major initiating step in the pathogenesis of vascular disease and atherosclerosis. The identification and quantification of circulating endothelial cells (CECs) has evolved as a novel marker of endothelial function. As a technique, it correlates with other markers of endothelial function such as flow-mediated dilation, the measurement of von Willebrand factor, and tissue plasminogen activator. Quantification of CECs is difficult due to low numbers, variable morphology, and a lack of standardization in current techniques used. CECs appear to be a different population of cells to endothelial progenitor cells. Increased CECs have been noted in a number of disease states and is evolving as a novel method of assessment of both disease severity and response to treatment. [ABSTRACT FROM AUTHOR]

Numerous and diverse experimental animal models have been used over the years to examine reactions to various forms of blood vessel disease and/or injury across species and in multiple vascular beds in a cumulative effort to relate these findings to the human condition. In this context, the rat carotid artery balloon injury model is highly characterized and commonly used for investigating gross morphological, cellular, biochemical, and molecular components of the response to experimentally induced arterial injury. The mechanical damage caused by the balloon catheter completely removes the intimal endothelial lining and creates a distending mural injury in the operated vessel. This elicits a reproducible remodeling response characterized by vascular smooth muscle cell (SMC) mitogenesis and migration (through phenotypic switching), SMC apoptosis, partial vascular endothelial cell regeneration, enhanced matrix synthesis, and establishment of an invasive neointima in time-dependent fashion. This multi-factorial process allows for investigation of these many important pathophysiological processes and can serve as a valuable ‘‘proof-of-concept'' tool to verify and substantiate in vitro results; however, inherent anatomical and adaptive constraints of this in vivo model ration comparison to the diseased human system (seeNote 1). In this chapter, brief overview of the materials needed and the methodologies commonly employed for successful routine performance of this important experimental animal model is provided. Individual sub-sections will cover animal care and handling, pre-operative and post-operative procedures, and the surgery proper. Protocols for histopathology and morphometry and procedures for data management and interpretation pertinent to the rat carotid artery balloon injury model are discussed in Chapter 2. [ABSTRACT FROM AUTHOR]

The integral role of apoptosis in the pathogenesis of cardiovascular diseases has been extensively studied and characterized in recent years. The study of cell death in the vasculature has significantly contributed to our knowledge of vascular disease pathology and has played a role in identifying potential therapeutic strategies for these diseases. This chapter describes a number of standard, widely used protocols for detecting and quantifying apoptosis in vessel wall cells and tissue. These techniques include terminal deoxynucleotidyl transferase dUTP nick-end labeling staining for DNA fragmentation, Hoechst staining for chromatin condensation, Annexin V staining, labeling for phosphatidylserine externalization, Western blot assessment of caspase cleavage, immunofluorescence detection of caspase activation, assessment of mitochondrial membrane depolarization and cytochrome c release, and a splenocyte assay for quantifying susceptibility to immune cell-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Renin angiotensin aldosterone system (RAAS) activation plays an essential role in the development of cardiovascular disease (CVD). Multiple pathophysiologic processes are able to activate RAAS, among which hypertension, obesity, diabetes mellitus 2, and chronic kidney disease deserve special attention, because they are the main contributors to CVD. Adding to the well-known effects of RAAS overactivity on the vasculature and water and electrolyte balance, current evidence links abnormal activation of the RAAS to increased production of reactive oxygen species (ROS) and oxidative stress. This association is mediated at least partially through interaction of angiotensin II (Ang II) with its receptor angiotensin receptor 1 (AT1R) in cardiovascular tissue, and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) enzymatic complex, which finally leads to increased ROS production. This resulting state of enhanced oxidative stress contributes largely to generalized atherosclerosis and finally to CVD. The generation of animal models of increased RAAS and Ang II expression, in particular the Ren2 rodent model, provides important opportunities to better characterize the relationship between this system and the production of ROS. This chapter describes methods to evaluate, characterize, and quantify the activity of the RAAS and NADPH oxidase, as well as the production of ROS production in animal model of RAAS. [ABSTRACT FROM AUTHOR]

Glucose metabolism plays an important role in cardiac bioenergetics that changes under various stress conditions including hypertrophy, diabetic cardiomyopathy, and ischemia-reperfusion injury. To understand the role of glycolysis under these conditions, we have altered several steps of the glycolytic pathway specifically in the heart. In this chapter, we describe methods used to produce cardiac-targeted transgenic mice and procedures for measuring various glucose metabolites including glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, and glycogen. Also, we describe methods for measuring glucose transport and glycolysis in perfused mouse hearts. Using these methods, we show that mice over-expressing cardiac-specific kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Mykd-PFK-2) show reduced glucose transport and reduced glycolysis when compared with control. The metabolites glucose-6-phosphate, fructose-6-phosphate, and glycogen were elevated, whereas fructose-1,6-bisphosphate was reduced in the transgenic Mykd-PFK-2 mouse hearts. [ABSTRACT FROM AUTHOR]

Increased interest in cardiac safety and renewed interest in drugs for treating myocardial ischemia and congestive heart failure have led to increased use of cardiovascular models. Unfortunately, many molecular or cell-based screens are not perfect predictors of activity in vivo. One rapid means to test "proof of principle" in hearts is the isolated or Langendorff perfused heart system. This allows direct measurement of cardiac contractile function and coronary flow without interference from changes in the systemic circulation. The setup basically is a heart whose coronary arteries are retrogradely perfused through the aortic root with heated and oxygenated buffer solutions. We will describe the constant pressure technique as this is the most commonly used and allows us to directly measure changes in coronary flow without changes in turgor noted with constant flow methods. Instead of a classical working heart preparation, we describe the use of a balloon properly fitted into the left ventricle to measure end diastolic and peak systolic pressure making this a much simpler and less expensive technique without loss of scientific quality. Such a setup can be used to measure effects of compounds or treatments on coronary flow, contractile function either in the normal or pathological states. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is the leading cause of death in the industrialized world. Diabetes is a major risk factor for cardiovascular disease. Unchecked diabetes can also lead to renal failure, blindness, heart attack, stroke, and amputation. The focus of this chapter will be to review the different mechanisms of insulin resistance (IR)/type 2 diabetes and various animal models used to study cardiac changes during these conditions. [ABSTRACT FROM AUTHOR]

Hypertension affects approximately 25% of adults and is a major risk factor for cardiovascular disease. Although there are currently adequate therapeutic options for humans with hypertension, the molecular mechanisms underlying hypertension are still relatively unknown. The generation of hypertensive animal models provides an excellent modality to not only study the pathophysiology but also test innovative therapeutics. This chapter describes the detailed methods that utilize the drinking water of rats to develop models of nitric oxide synthase (NOS) inhibition-induced, guanosine triphosphate cyclohydrolase (GTPCH) inhibition-induced, and glucocorticoid-induced hypertension. [ABSTRACT FROM AUTHOR]

In response to an increased afterload, the myocardium undergoes a complex adaptation by which wall stress is normalized and cardiac output is maintained. Although the consensus suggests that the increase of the myocardial mass is a necessary adaptive process to accommodate the increased workload, there is growing evidence that hypertrophy ultimately results in pathological remodeling and deterioration of cardiac function. Despite intense investigation, our understanding of the cellular mechanisms that are responsible for the initiation and the maintenance of this adaptation is largely incomplete and preventing or regressing left ventricular hypertrophy (LVH) is a major challenge. This chapter provides a detailed description of the procedures necessary to induce LVH by coarctation of the transverse aorta and to analyze the effects of the increased hemodynamic load on cardiac mass, cardiomyocyte size, and cardiac performance. [ABSTRACT FROM AUTHOR]

Atherosclerotic lesions develop through interactions with diverse cell types whose functions are determined by a complex array of regulators. Immunostaining is now a commonly applied technique to identify these numerous cell types and regulators in lesions. The principle of the technique is that an antibody is incubated with the tissue under conditions that favor a specific interaction with its antigen. This is subsequently visualized most commonly by a chromogenic substrate that produces a colored precipitate at the location of the antigen-antibody interaction. When appropriately applied, it is a powerful technique to provide mechanistic insight into the atherogenic process. However, the complexity of atherosclerotic tissue can provide challenges to ensuring that development of the chromogen is due to specific antigen-antibody interaction. Thus, the determination of specific interactions frequently requires the judicious use of appropriate control experiments. [ABSTRACT FROM AUTHOR]

It is widely believed that rupture of a vulnerable atherosclerotic plaque leads to acute coronary events and stroke. However, the exact mechanisms involved in the plaque rupture remain unknown. Pathological animal models are valuable in the research on human disease mechanism, their therapy, and drug development. Recently, we have proposed a novel murine model of atherosclerotic plaque rupture associated with not only intraplaque hemorrhage but also luminal thrombus using combination treatments of ligation and perivascular cuff placement on the carotid artery. [ABSTRACT FROM AUTHOR]

Experiments aimed at analyzing the response of blood vessels to mechanical injury and ensuing remodeling responses often employ the highly characterized carotid artery balloon injury model in laboratory rats. This approach utilizes luminal insertion of a balloon embolectomy catheter into the common carotid artery with inflation and withdrawal resulting in an injury characterized by vascular endothelial cell (EC) denudation and medial wall distension. The adaptive response to this injury is typified by robust vascular smooth muscle cell (SMC) replication and migration, SMC apoptosis and necrosis, enhanced synthesis and deposition of extracellular matrix (ECM) components, partial vascular EC regeneration from the border zones, luminal narrowing, and establishment of a neointima in time-dependent fashion. Evaluation of these adaptive responses to blood vessel injury can include acute and longer term qualitative and quantitative measures including expression analyses, activity assays, immunostaining for a plethora of factors and signals, and morphometry of neointima formation and gross mural remodeling. This chapter presents a logical continuation of Chapter 1 that offers details for performing the rat carotid artery balloon injury model in a standard laboratory setting by providing commonly used protocols for performing histological and morphometric analyses in such studies. Moreover, procedures, caveats, and considerations included in this chapter are highly relevant for alternative animal vascular physiology/pathophysiology studies and in particular those related to mechanisms of vascular injury and repair. Included in this chapter are specifics for in situ perfusion-fixation, tissue harvesting and processing for both snap-frozen and paraffin-embedded protocols, specimen embedding and sectioning, slide preparation, several standard histological staining steps, and routine morphological assessment. [ABSTRACT FROM AUTHOR]

Mice expressing the KRN T cell receptor transgene and the MHC class II molecule Ag7 (K/B×N mice) develop severe inflammatory arthritis, and serum from these mice causes similar arthritis in a wide range of mouse strains, owing to pathogenic autoantibodies to glucose-6-phosphate isomerase (GPI). This model has been useful for the investigation of the development of autoimmunity (K/B×N transgenic mice) and particularly of the mechanisms by which anti-GPI autoantibodies induce joint-specific imflammation (serum transfer model). In this chaper, after a summary of findings from this model system, we describe detailed methods for the maintenance of a K/B×N colony, crossing of the relevant TCR and MHC genes to other strain backgrounds, evaluation of KRN transgenic T cells, measurement of anti-GPI antibodies, induction of arthritis by serum transfer, and clinical and histological evaluation of arthritis. [ABSTRACT FROM AUTHOR]

Fibrocytes circulate in the peripheral blood, produce collagen and other matrix proteins, and express cell surface markers indicative of a hematopoetic origin distinguishing them from fibroblasts. Circulating fibrocytes were first identified in 1994 in a model system of wound repair, and defined by their growth characteristics and unique surface phenotype. The methods currently employed for the isolation, growth, and characterization of peripheral blood fibrocytes rely on the entry of "fibroblast-like" cells into wound chambers, or the derivation of "fibroblast-like" cells from the buffy coat of peripheral blood obtained from different mammalian species. In this protocol, we culture fibrocytes from the mononuclear cells of peripheral blood and harvest the cultured cells for flow cytometry analysis. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis is characterized by inflammation of the joints and degradation and invasion by fibroblast-like synoviocytes (FLS) of the cartilage. To assess the invasiveness of FLS an in vitro invasion assay was developed. In this invasion assay the FLS grow through an artificial matrix composed mainly of collagen IV. First, the walls of transwells are coated with paraffin to avoid meniscus formation. Subsequently, the bottoms of the transwells (on top of the membrane) are coated with a thin layer of matrigel. On top of this matrigel fibroblast-like synoviocytes are seeded at a density of 100,000 cells per milliliter. The cells are cultured in serum free medium in the inner compartment inside the transwell. To the outer compartment outside the transwell IMDM with 10% fetal calf serum and 10% NHS is added. The cells are incubated for 3 d at 37°C and 5% CO2. After 3 d the cells are fixed with 2% glutaraldehyde in phosphate buffered saline and stained with 1% crystal violet in water. The matrix on the inside of the transwells and the cells that have not grown through the matrix and the membrane are removed. The cells that have grown through the matrix and through the membrane under the transwell can be visualized by light microscopy and counted. [ABSTRACT FROM AUTHOR]

The use of gene transfer techniques has become of utmost importance both for the analysis of molecular pathways of rheumatic joint destruction and for the evaluation of novel therapeutic concepts to treat rheumatic diseases. However, gene transfer into synovial fibroblasts faces several challenges, which result mainly from the lack of specific surface markers and the low-proliferation rate of these cells. This chapter describes both nonviral and viral strategies of transferring gene constructs into synovial fibroblasts. It focuses on the use of lipofection for the gene transfer of siRNA to synovial fibroblasts and the use of AMAXA-nucleofection for the nonviral transfer of gene expression constructs. In addition, retro- and lentiviral strategies of gene transfer are introduced. Finally, the SCID mouse in vivo model of rheumatoid joint destruction is described as a means of evaluating the effects of gene transfer on the invasiveness of synovial fibroblasts. [ABSTRACT FROM AUTHOR]

Evaluation of differentially regulated genes is essential for the development of novel therapeutic approaches in multifactorial diseases such as rheumatoid arthritis (RA). RA synovial fibroblasts (RASF) are key players in inflammation and cartilage destruction. Therefore, RASF are important cellular targets for the analysis of gene expression profiles. Such analyses may include a comparison of SF from nontreated RA patients with those from treated RA patients, and may also be used to evaluate which genes and intracellular processes can be modulated through genetic modification, gene transfer, and drug treatment for the identification of the pathways driving the destructive behavior of these cells. This chapter reports the combination of RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and cDNA array with defined genes for a highly sensitive analysis of gene expression profiles in RASF using small amounts of total RNA. RNA can be extracted from cultured SF, isolated, and analyzed using RAP-PCR with different arbitrary primers for first- and second-strand synthesis to generate a radioactive labeled probe which can be used for cDNA array hybridization. Visualization and evaluation of gene expression can be performed by phosphorimaging in combination with an array-specific software analyzing system followed by statistic evaluation of the generated data. In summary, the combination of RAP-PCR combined with cDNA arrays is a sensitive method to identify differentially expressed genes in RASF with high specificity, especially for low abundant mRNAs. [ABSTRACT FROM AUTHOR]

The study of fibroblast-like synoviocytes (FLS) has yielded important insights into the pathogenic mechanisms of rheumatoid arthritis. FLS can be cultured from synovial tissue obtained at joint replacement surgery, synovectomy, or synovial biopsy. After collagenase digestion, adherent cells consist mainly of synovial fibroblasts and synovial macrophages. Proliferating FLS are enriched by repeated passage and comprise >95% of cells by passage 3. Because of cell senescence, use of FLS lines after passage 9 is generally not recommended. FLS in culture have a distinct phenotype with regard to morphology, ultrastructure, surface phenotype, and function. Surface markers that can be used to characterize FLS include positive staining for VCAM-1, CD44, CD55, CD90 (Thy-1), and cadherin-11, coupled with the absence of macrophage markers such as CD14 or CD68. [ABSTRACT FROM AUTHOR]

The ingress of leukocytes into the synovium is a key event in inflammatory arthritis, such as rheumatoid arthritis. A number of soluble inflammatory mediators, such as chemokines, cytokines, and soluble adhesion molecules are involved in this event. It is feasible to test the chemotactic activity of mononuclear cells or endothelial cells using in vitro chemotaxis assays. The role of various chemotactic mediators in inflammatory synovitis can also be assessed using these systems by including these agents in the assay. There we describe monocyte and endothelial cell chemotaxis assays used in our laboratory. [ABSTRACT FROM AUTHOR]

Angiogenesis is the formation of new blood vessels from existing vessels. The formation of new vessels appears to be an early and fundamental process for the evolution of the inflammatory response in synovial joints affected by arthritis. The propagation of new vessels in the synovial membrane allows the invasion of this tissue over the intra-articular cartilage in an adherent fashion. This process appears to support the active infiltration of synovial membrane into cartilage and results in erosion and destruction of the cartilage. This process results in joint damage and ultimately in deformity, as the normal joint architecture and balance of tendons becomes disrupted. Angiogenesis may be assessed in vivo by direct visualization through the introduction of a needle arthroscope using local anesthesia, differential patterns of vascular morphology have been described in seropositive rheumatoid arthritis and seronegative arthritides such as psoriatic and reactive arthritis. At a microscopic level, angiogenesis may be examined in the tissue sections using immunohistochemistry or immunofluorescence. Endothelial cells may also be studied in vitro in culture to examine production of angiogenic growth factors, cell activation, migration, and tubule formation. Finally, synovial biopsy explants may be cultured ex vivo to provide a model simulating the intra-articular milieu. [ABSTRACT FROM AUTHOR]

A complete pattern of adhesion molecules and chemokines involved in leukocyte migration in different tissues and in homeostatic vs inflammatory conditions is still lacking. This chapter describes how to characterize the mechanisms of leukocyte recruitment in synovial vessels in vivo by using epifluorescence videomicroscopy. It has been proposed that inflamed endothelium expresses a combination of adhesion ligands and activating factor(s) for Gi-linked receptors that together orchestrate leukocyte recruitment in vivo. Thus, the combination of molecules involved in the adhesion cascade in synovial vessels may favor the arrest of specific leukocyte subpopulations during different phases of joint inflammation. The approach presented here may provide a useful tool for further investigations of physiologic and pathologic events that occur in normal synovial microcirculation as well as during arthritis. [ABSTRACT FROM AUTHOR]

To be able to visualize real time leukocyte — endothelial cell interactions in vitro opens up the possibility of exploring the complex cascade of events that culminate in leukocyte recruitment and diapedesis in a much more detailed and controlled way. Techniques have been developed whereby fluorescently labeled leukocytes are perfused over an endothelial substrate in a controlled manner. Interactions can then be visualized and, using motion tracking software, the movement of cells characterized. Dynamic flow based adhesion assay protocols build on previous static assays of leukocyte adhesion in better modeling the environment in which these interactions actually take place in vivo. [ABSTRACT FROM AUTHOR]

The in vitro isolation, propagation and study of endothelial cells (EC) is an invaluable means by which the function of the vascular endothelium in physiology and pathophysiology can be explored. In recent years heterogeneity between large and small vessel EC, between arteries and veins, and between microvascular EC derived from different organs has become increasingly apparent. This has led to the development of protocols for the isolation of these different EC. In addition, the data emerging on vascular EC function in gene-targeted mice has highlighted the need for reliable methods of isolation of murine EC. This chapter describes methods for the isolation, characterization and culture of macro- and microvascular EC from a variety of species and introduces simple approaches to investigating their surface antigen expression. [ABSTRACT FROM AUTHOR]

Osteoclasts are bone-resorbing multinucleated cells derived from the monocytemacrophage lineage. The authors have developed a mouse marrow culture system and a coculture system of mouse osteoblasts and hemopoietic cells, in which osteoclasts are formed in response to various osteotropic factors such as 1α,25-dihydroxyvitamin D3, parathyroid hormone, prostaglandin E2, and interleukin -11. Recent studies have revealed that osteoblasts express two cytokines essential for osteoclastogenesis: receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Using RANKL and M-CSF, we can induce osteoclasts from monocytemacrophage lineage cells even in the absence of osteoblasts. This chapter describes the methods for osteoclast formation in vitro in the presence and absence of osteoblasts, and for pit-formation assay using dentine slices and osteoclasts formed in vitro. These culture systems have made it possible to investigate each step of osteoclast development and function separately. [ABSTRACT FROM AUTHOR]

Rodent arthritis models are tools to study joint destruction. This chapter describes methods to prepare and analyze histological sections from inflamed paws. Protocols to create decalcified paraffin-embedded as well as undecalcified plastic-embedded sections are provided. Strengths and limitations of these two histological techniques are described. Major staining techniques on hard tissue sections focusing on the evaluation of joint destruction are indicated. Finally, evaluation of arthritic paw sections by histomorphometry, a semiautomated computerized method to measure areas of interest, is described. [ABSTRACT FROM AUTHOR]

This chapter provides practical information on the assay of tissue inhibitor of metalloproteinase (TIMP) activity and background information enabling meaningful interpretation of the data. Protocols are given for assessing the presence of TIMPs in biological samples by immunoblotting and by virtue of their ability to inhibit matrix metalloproteinase (MMP) hydrolysis of protein substrates (reverse zymography) and synthetic fluorogenic substrates. [ABSTRACT FROM AUTHOR]

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane protein which exhibits biological activities on the cell surface. One of the characteristic functions of this enzyme is activation of proMMP-2 on the cell surface (1). This process can be monitored using gelatin zymography to detect the pro- and active forms of MMP-2. Cellular MT1-MMP activity can also be directly detected by in situ degradation of layers of either gelatin or collagen. By combining inhibitors, western blotting, and immunohistochemistry, one can detect and conclude the presence of MT1-MMP activity in the cells. [ABSTRACT FROM AUTHOR]

Two methods for the assessment of the expression of gelatinases A and B, MMP-2 and MMP-9, in articular cartilage are described. Immunohistochemical analysis of tissue sections provides information about the precise localization of the enzymes within the tissue, pinpointing the cells that synthesize the proteinases, and zymography of cell/tissue conditioned culture media allows a semi-quantitative assessment of the gelatinases and their activation status. [ABSTRACT FROM AUTHOR]

Assay of collagenase activity involves the use of radiolabeled collagen. Stimulation of cartilage with proinflammatory cytokines results in the upregulation of collagenases and the subsequent release of degraded collagen fragments. These enzymes can be localized in both osteoarthritic and rheumatoid arthritis cartilage and synovial tissues. [ABSTRACT FROM AUTHOR]

Quantitation of glycosaminoglycans (GAGs) in the form of aggrecan fragments released from cartilage in culture is a simple way to determine the efficacy of different cytokines alone or in combination in simulating cartilage catabolism. Two approaches for GAG assay are described, with special attention being paid to the advantages and limitations of each method [ABSTRACT FROM AUTHOR]

Necrosis and apoptosis have been demonstrated in articular cartilage in response to trauma and disease. However, cell death in articular cartilage may also be thought of as a scale of cell death culminating in secondary necrosis with the failure to remove apoptotic cells from the tissue. The in situ detection of cell death is an important technique in studying articular cartilage as it most closely resembles the in vivo situation. The methods described here involve the use of light microscopy and electron microscopy in conjunction with fluorescent and biochemical methods to correctly ascertain the type of cell death that has occurred. [ABSTRACT FROM AUTHOR]

Many studies in arthritis research require an evaluation of the cellular responses within the joint and the ensuing matrix degradation in articular cartilage. The early histochemical/histological scale of Mankin (1) has been widely used but recently challenged as insufficient (2). Imaging techniques such as microscopic magnetic resonance imaging (MRI) (3), polarized light microscopy (3), atomic force microscopy (4), and infrared spectral analysis (5) have opened new approaches to evaluating cartilage structure. Histological methods now include in situ hybridization for cell-specific gene expression and immunohistochemistry for the spatial organization of cartilage proteins and their processed forms. This chapter details of a method for immunohistochemical analysis of aggrecan degradation in articular cartilage samples which have been prepared by standard methods of formalin fixation and paraffin embedding. The procedure focuses on the application of antibodies (e.g., anti-ADAMTS4, anti-MT4MMP) which detect some of the proteinases most likely involved, and anti-NITEGE which detects the terminal product of the aggrecanase-mediated cleavage of aggrecan at Glu392-Ala393 (bovine, human, dog, rat, pig, sheep, horse, mouse) or Glu393-Ala394 (chick). [ABSTRACT FROM AUTHOR]

In rheumatology and joint research, as in other fields, a purely descriptional appqoach to morphology cannot satisfy the exactions of modern clinical medicine. Investigators now appreciate the need to gauge pathological changes and their response to treatment by quantifying susceptible structural parameters. But the desired information respecting three-dimensional structures must be gleaned from either actual or virtual two-dimensional sections through the tissue. This information can be obtained only if the laws governing stereology are respected. In this chapter, the stereological principles that must be applied, and the practical methods that have been devised, to yield unbiased estimates of the most commonly determined structural parameters, namely, volume, surface area and number, are summarized. [ABSTRACT FROM AUTHOR]

Quantitative image analysis is a form of imaging that includes microscopic histological quantification, video microscopy, image analysis, and image processing. Hallmarks are the generation of reliable, reproducible, and efficient measurements via strict calibration and step-by-step control of the acquisition, storage and evaluation of images with dedicated hardware and software. Major advantages of quantitative image analysis over traditional techniques include sophisticated calibration systems, interaction, speed, and control of inter- and intraobserver variation. This results in a well controlled environment, which is essential for quality control and reproducibility, and helps to optimize sensitivity and specificity. To achieve this, an optimal quantitative image analysis system combines solid software engineering with easy interactivity with the operator. Moreover, the system also needs to be as transparent as possible in generating the data because a "black box design" will deliver uncontrollable results. In addition to these more general aspects, specifically for the analysis of synovial tissue the necessity of interactivity is highlighted by the added value of identification and quantification of information as present in areas such as the intimal lining layer, blood vessels, and lymphocyte aggregates. Speed is another important aspect of digital cytometry. Currently, rapidly increasing numbers of samples, together with accumulation of a variety of markers and detection techniques has made the use of traditional analysis techniques such as manual quantification and semi-quantitative analysis unpractical. It can be anticipated that the development of even more powerful computer systems with sophisticated software will further facilitate reliable analysis at high speed. [ABSTRACT FROM AUTHOR]

In rheumatoid arthritis (RA), the synovium represents the predominant site of inflammation and joint destruction and is regarded as the key organ involved in disease pathogenesis. It has been studied in different ways over the last 30 yr, yielding information about the mechanisms involved in disease and remains the tool most proximal to understanding the pathogenesis of RA. This chapter outlines how both histological and in vitro studies of dissociated tissue played key roles in the development of biological anti-TNF-α therapy and provides detailed protocols used routinely in the laboratory to facilitate studies of RA synovium and its composite cell populations. [ABSTRACT FROM AUTHOR]

Most current approaches used to analyze gene expression in tissue samples are based on RNA isolated either from cultured synovial cells or from synovial biopsies. However, this strategy does not distinguish between specific gene expression profiles of cells originating from discrete tissue areas. Therefore, we established the combination of laser-mediated microdissection and RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display to analyze profiles of gene expression in histologically defined areas of arthritic tissue. Cryosections derived from synovial tissue were used to obtain cell samples from different tissue areas of both rheumatoid arthritis (RA) and osteoarthritis (OA) patients using a microbeam laser microscope. RNA was isolated and analyzed using nested RNA arbitrarily primed PCR to generate a fingerprint of the expressed gene sequences. Differentially expressed bands were isolated, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry. [ABSTRACT FROM AUTHOR]

Subtractive hybridization, like serial analysis of gene expression (SAGE), RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and microarrays, is a screening method for differentially expressed genes. At first, poly-A+-RNA is isolated and reverse transcribed into cDNA. With the SMART technology, total RNA can be used. For subtractive hybridization, two adaptors are ligated to the tester cDNA. The tester cDNA is then mixed twice with cDNA of the reference sample. Sequences that are present in equal levels in the tester and in the reference cDNA hybridize to each other and are then removed. In contrast, differentially expressed genes are highly enriched and then amplified by PCR. Because subtractive hybridization is a complex multistep procedure, the results should be controlled at each level and positive controls are recommended. Because all screening methods produce a significant number of false positives, the differential expression has to be confirmed by independent methods. [ABSTRACT FROM AUTHOR]

The chapter focuses on the detection of specific mRNA by in situ hybridization (ISH) in synovial tissue specimens. This technique is widely applied, reliable, specific, and sensitive, because even small quantities of mRNA can be detected. Presented here contemporary protocols for ISH using a combined nonradioactive immunohistochemical detection system. In overview, the following steps have to be covered to perform ISH. (1) mRNA probes (sense and antisense) are generated by in vitro transcription of cDNA utilizing digoxigenin-labeled UTP nucleotides, (2) fixed tissue sections are digested with trypsin and treated consecutively with prehybridization solutions, (3) hybridization with labeled riboprobes takes place at 50°C overnight in a humid chamber, (4) unbound riboprobe is removed by incubation with RNase A and additional washing with buffers, (5) stringent washing steps are performed with solutions of different sodium dodecyl sulfate, SSC, and formamide concentrations, (6) digoxigenin-labeled probes are detected immunohistochemically using antidigoxigenin antibodies linked with alkaline phosphatase and NBT/BCIP as detection system. [ABSTRACT FROM AUTHOR]