Laser microdissection (LMD) is an accurate and fast method to procure pure populations of cells from complex, heterogeneous tissues under direct microscopic visualization. It can be applied to a wide range of cell preparation, including paraffin-embedded material. The morphology of the captured cells is retained, and DNA, RNA, and proteins can be extracted for molecular analysis. The potential applications of LMD to human cardiovascular research are multiple, including viral/autoimmune myocarditis and arteritis, atherosclerotic lesions, and myocardial and vascular cell proliferation and death. Molecular and genetic analysis of LMD-procured cells in cardiac and vascular tissues may provide a better understanding of several cardiac diseases.
Key words: cardiovascular diseases; endomyocardial biopsy; inflammatory cardiomyopathy; laser microdissection; myocarditis
Abbreviations: EBV = Epstein-Barr vires; LMD = laser micredissection; mRNA = messenger RNA; mtDNA = mitochondrial DNA; PCR = polymerase chain reaction
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A growing number of genomic and proteomic techniques have become available to assess messenger RNA (mRNA) transcripts and proteins in human cells and tissues. In particular, the differential expression of these molecules between normal and pathologic tissues can provide indications for specific mechanisms of diseases. However, as tissues are usually composed of heterogeneous cell populations, the average information obtained from the molecular analysis of biological samples as a whole may be of limited value. To address this difficulty, several manual or automatic methods have been developed for selecting specific cell populations from a given tissue sample. Among these methods, laser microdissection (LMD) provides considerable advantages and can be successfully applied to human cardiovascular research.
LASER MICRODISSECTION: METHODOLOGIC ASPECTS
LMD is a technique that enables the procurement of homogeneous types of cells from both frozen and fixed tissue sections and from cytologic smears, using a laser integrated into a standard microscope. It can be routinely utilized to collect pure populations of targeted cells for subsequent DNA, RNA, and protein analysis. Although the initial cost for the microscope and accompanying computer hardware and software is quite high, LMD has the advantages of being user friendly, precise, rapid, and guarantees the purity of the collected material, which is not feasible using previous manual microdissection techniques. Compared to LMD, in situ techniques such as in situ hybridization and in situ polymerase chain reaction (PCR) (1-2) are time consuming and technically complex, while immunohistochemistry lacks comparable sensitivity and specificity and is not suitable for nucleic acid analysis (Table 1).
Several systems are available for LMD, and vary in cell-capture method and system configuration (Table 2). The first LMD system, developed in the mid-1990s as a research tool at the National Institutes of Health, uses a fusion technique for cell capture. Laser capture microdissection is based on the selective adherence of targeted cells and tissue fragments to a thermoplastic membrane, mounted on a transparent cap and activated by a low-energy infrared laser pulse. (3,7,8) The thermoplastic film lies directly on the surface of the tissue section, mounted on a glass slide, and contains special infrared absorbing dyes that melt and fuse with the selected underlying cell type when the laser is activated. When the film is removed, the selected cells remain bound to the film, leaving behind the remaining tissue. The tissue suffers only brief thermal transients that do not damage DNA, RNA, or proteins. Importantly, this method can be applied also to stained, archival glass slides.
The other widely used system is the laser microbeam microdissection system that uses a pulsed ultraviolet laser with a small beam to cut out the cells of interest by photoablation of the adjacent tissue. (9,10) Tissue sections are mounted on a thin polyethylene foil slide, and the cells of interest are microdissected by means of an ultraviolet laser that performs the circumferential dissection of the selected cells following a precisely drawn incision path (Fig 1). By this cold ablation, the material to be extracted is not directly exposed to the laser. The microdissected tissue areas are then collected on the cap of a nanotube for subsequent molecular analysis, using contamination and contact-free methods of cell recovery.
[FIGURE 1 OMITTED]
When the cells of interest exhibit characteristic disease morphology, LMD can be carried out on hematoxylin-eosin-stained tissue sections (Fig 1). However, in order to select cell types that are not readily distinguished by morphologic examination, the expression of specific cell markers by immunohistochemically guided procedures may be employed.
In both the laser capture and laser microbeam microdissection systems, the laser is integrated into a standard microscope. The investigator can examine the tissue section microscopically before and after the microdissection procedure and control the homogeneity of the selected material, as the tissue collected on the cap retains its original morphology. These systems also offer the possibility to archive the images of each dissection, allowing the correlation of the histopathologic findings with the subsequent molecular results.
APPLICATION OF LMD TO CARDIOVASCULAR RESEARCH
Up to now, the major field of application of this method has been cancer research. Separation of malignant, in situ, and normal cell subpopulations within a single biopsy specimen allows a direct comparison of tissue DNA, RNA, and protein content and function in both normal and neoplastic tissues. (11) This technique has been applied to detect both known and unknown alterations in the genomes of a variety of tumors. (12) Moreover, microarray technology has been used to obtain molecular fingerprints of gene expression in microdissected tissue biopsies of human cancer, contributing to identify different disease stages and specific molecular-targeted therapies. (13)
Recently, this method has been applied to human cardiac and vascular tissue (Table 3). Moniotte et al (14) used LMD to selectively capture myocytes and endothelial cells from cardiac frozen sections stained with hematoxylin-eosin. The authors quantitatively assessed the expression of rare mRNAs (ie, [[beta].sub.3]-adrenoceptor mRNAs) in the small number of collected cells using real-time PCR, and demonstrated the efficacy of combining these two methods for gene expression studies in cardiac diseases. Similarly, De Souza et al (15) performed LMD on frozen left and right ventricular tissue from explanted hearts to selectively collect blood vessels and myocytes, and analyzed the protein extracts by two-dimensional gel electrophoresis. Their results showed good protein recovery from the microdissected cells and well-distinct protein profiles, demonstrating the feasibility of using LMD to perform proteomic studies of individual cardiac tissue components. LMD has been also employed to analyze the level of deleted mitochondrial DNA (mtDNA) in the conducting myocardium in comparison with working myocarchum in a patient with Kearns-Sayre syndrome, (16) and showed a greater abundance of mutated mtDNA in the conducting system, consistent with its prevalent clinical involvement.
More recently, the LMD technique was applied to endomyocardial biopsy specimens in order to precisely separate and collect the cardiomyocytes and the infiltrating lymphocytes in the cardiac tissue infected by Epstein-Barr virus (EBV). (17) Paraffin-embedded sections from nine patients with chronic heart failure and a histologic diagnosis of inflammatory cardiomyopathy, with PCR positivity for EBV on whole frozen tissue, were studied. LMD was applied in this series to assess whether the viral genome was localized in the myocytes, the inflammatory cells, or both. The selection of cells was immunohistochemically guided by [alpha]-sarcomeric actin antibody staining for cardiomyocytes and CD45RO staining for lymphocytes (Fig 2). EBV genome was detected by PCR analysis in cardiomyocytes of all patients and was absent in infiltrating lymphocytes, thus suggesting a cytopathic role of this virus and the opportunity for an antiviral/immunomodulatory therapy. Importantly, this study showed that PCR analysis of nucleic acids extracted from a small number of microdissected cells does not cause a loss of sensitivity compared with PCR performed on the whole frozen tissue, with the advantage to allow the cellular localization of the viral agent. The combination of highly sensitive PCR analysis with the microscopical selection of the targeted cells might be useful in the investigation of the different cellular tropism (ie, cardiomyocytes, endothelial cells, fibroblasts, smooth muscle cells) of the viruses infecting the heart, thus improving our knowledge of viral myocarditis in terms of pathogenesis, clinical manifestations, and possible therapeutic options. Similarly, this method can be applied to human atherosclerotic lesions to investigate the presence of microbial agents in the different cellular components of the plaque (ie, foam cells, smooth-muscle cells, lymphocytes, endothelial cells, fibroblasts).
[FIGURE 2 OMITTED]
Tuomisto et al (18) used LMD to isolate macrophage-rich shoulder areas from human atherosclerotic lesions and compared their gene expression patterns to macroseopically normal diffuse intimal thickening by complementary DNA array. The authors identified several overexpressed genes, such as hydroxymethylglutaryl coenzyme A reductase, colony-stimulating factor receptors, integrin receptors, and nitric oxide synthase, and suggested the usefulness of this approach to identify therapeutic targets for the treatment of atherosclerotic diseases.
In another study, Martinet et al (19) used LMD to isolate caspase-2--positive macrophage-derived foam cells around the necrotic core of atherosclerotic plaques in human carotid endoarterectomy specimens. The authors analyzed, using Western blotting, the proteins extracted from the targeted cells and identified the overexpression of a 35-kd protein (caspase-2S) with antiapoptotic effect, suggesting that the upregulation of this protein in macrophages can represent a mechanism to survive the increased levels of oxidative DNA damage present in the atherosclerotic plaque. LMD can also be useful to investigate inflammatory diseases of unknown etiology and uncertain treatment, involving the heart and/or the vessels, such as giant-cell myocarditis and arteritis.
Giant cell myocarditis is a disease of young, predominantly healthy adults, frequently with a fatal course. (20) Its etiology is unknown; it may recur after cardiac transplantation and sometimes responds to immunosuppressive therapy. Histologically, it is characterized by extensive inflammatory infiltrates with the presence of abnormal multinucleated cells, ie, giant cells, of possible myogenic or macrophagic origin. As illustrate in Figure 1, giant cells can be easily dissected from the endomyocardial tissue sample, and molecular analysis of the isolated material can give important information on the knowledge of this heterogeneous disease, providing indications for its treatment that are not established. Similarly, Gordon et al (21) used LMD to isolate giant cells from inflammatory lesions of giant cell arteritis and identified the presence of microbial sequences in the DNA extracted from the collected material, showing that this disease, considered autoimmune in nature, may be associated with a microbial infection.
Finally, the fact that the heart is no longer a postmitotic organ and that myocardial regeneration occurs in adult human heart opens a new potential field for application of LMD. Indeed, studies (22,23) on the chimerism of the heart after sex-mismatched transplantation have provided consistent results concerning the migration of primitive cells from the host to the graft, showing the presence of Y chromosome in myocytes of female hearts transplanted into male recipients. A population of resident cardiac primitive cells able to differentiate in myocytes, smooth-muscle cells, and endothelial cells has been identified in the adult human heart, and it has been shown that cardiac regeneration is highly activated in acute pathologic conditions, while the regenerative capacity decreases in the chronically decompensated heart and in aged diseased hearts. (24,25) LMD may offer the possibility to selectively study the newly generated myocytes as well as the resident cardiac primitive/ progenitor cells, recognized by specific cell markers. In addition, LMD allows the investigation of the expression of genes that can activate primitive cell growth and differentiation or inhibit cell replication and promote telomere erosion and primitive cell death in the acutely decompensated human heart, as well as in chronic heart failure. In conclusion, LMD is an extremely useful tool that can open new areas in the study of several cardiac diseases, combining advanced molecular biology techniques with the fast and precise selection of targeted cells under direct microscopic visualization of cardiac and vascular tissues.
Manuscript received February 7, 2005; revision accepted April 11, 2005.
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Cristina Chimenti, MD, PhD; Maurizio Pieroni, MD, PhD; Andrea Russo, MD, PhD; Patrizio Sale, MD; Matteo A. Russo, MD; Attilio Maseri, MD; and Andrea Frustaci, MD, FCCP
* From the Cardiothoracic and Vascular Department (Drs. Chimenti, Pieroni, and Maseri), Vitae Salute University, Milan; Department of Cardiology, Catholic University (Dr. Frustaci), Rome; Biopathology and Diagnostic Imaging Department (Dr. A Russo), "Regina Elena" Institute, Rome; and Pathology and Experimental Medicine Department (Drs. Sale and M. Russo), "La Sapienza" University, Rome, Italy.
Correspondence to: Andrea Frustaci, MD, FCCP, Cardiology Department, Catholic University, Largo A Gemelli 8, 00168, Rome, Italy; e-mail: biocard@rm.unicatt.it
COPYRIGHT 2005 American College of Chest Physicians
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