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Vascular and Interventional Radiology |
1 From the Departments of Radiology (L.F., J.P.S.) and Surgery (D.M.S.), Columbia University, 177 Ft Washington Ave, New York, NY 10032. Received June 17, 1998; revision requested August 14; revision received September 23; accepted February 12, 1999. Address reprint requests to L.F. (e-mail: lf66@columbia.edu).
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Abstract |
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 TOP Abstract IntroductionMATERIALS AND METHODSRESULTSDISCUSSIONAppendix 1References |
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MATERIALS AND METHODS: Coaxial curved stainless-steel guide wires were used to obtain samples of endothelial cells from large arteries and veins in 29 patients undergoing routine endovascular procedures. Three immunocytochemical markers were used to identify cells as endothelial. Cellular viability was evaluated in terms of cell membrane integrity, energy-dependent uptake of acetylated low-density lipoprotein, and cellular response to lipopolysaccharide. Single-cell reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study endothelial gene expression.
RESULTS: Cells with endothelial morphology and immunoreactivity for von Willebrand factor, thrombomodulin, and angiotensin-converting enzyme were consistently obtained from iliac and carotid arteries and large veins (average yield [± standard error] from 26 iliac arteries, 262 endothelial cells ± 45, 20%–30% of which were viable). These cells displayed induction of E-selectin messenger RNA at PCR after exposure to lipopolysaccharide. Expression of vascular cell adhesion molecule 1 transcripts in endothelial cells increased with patient age (P < .01), whereas expression of intercellular adhesion molecule 1 did not.
CONCLUSION: Viable endothelium can be obtained during routine angiography. Immunocytochemical and reverse transcription PCR analyses of these cells allowed determination of transcripts and proteins expressed by endothelium at sites of vascular lesions. Such information could aid in understanding mechanisms of vascular diseases and in clinical decision making.
Index terms: Arteries, biopsy, 9*.126, 9*.922 • Arteries, endothelium, 9*.922 • Arteries, iliac, 984.1261 • Biopsies, technology • Carotid arteries, 90.1261 • Molecular analysis, 9*.922 • Venae cavae, interventional procedure, 946.1261 • Pulmonary arteries, 944.1261
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Introduction |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONAppendix 1References |
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Such a view of healthy endothelium as a participant in a myriad of physiologic processes has led to the hypothesis that endothelial dysfunction may, by way of analogy, orchestrate pathophysiologic perturbation of the vasculature in disease states. Induction of endothelial expression of inflammatory mediators such as cytokines (10) and cell adhesion molecules (11) in the earliest phases of the inflammatory response and at lesion-prone areas, with respect to atherogenesis, supports the potential of the endothelial monolayer to regulate processes within the luminal vascular space, as well as processes at sites of tissue injury.
Insights into the properties of the endothelium have come from several lines of inquiry. Results from tissue culture experiments have demonstrated potential endothelial activities that are relevant in vivo, such as expression of inducible cell adhesion molecules (12). Authors of studies of vessel segments have emphasized particular properties of endothelialized versus deendothelialized vessels, as in the elaboration of nitric oxide (13). Investigators who have performed in vivo experiments with pathologic samples and, more recently, genetically manipulated mice have emphasized potentially pathogenic roles of key mediators such as vasoactive factors (14).
A critical missing link in the elucidation of endothelial mechanisms that contribute to vascular function in the normal and diseased vessel wall is the lack of information about the state of viable endothelial cells in situ; for example, their properties in healthy vessels and at the site of a lesion. Such a gap in our knowledge is due to the difficulty in obtaining viable endothelium from the vasculature in vivo.
These considerations led us to design a nondestructive method of in situ endothelial sampling by using an endovascular catheter. During endovascular procedures, catheters and guide wires inevitably come into contact with the vessel wall. We have found that endothelial cells become adherent to these vascular devices. A method was established to detach viable endothelial cells from the surface of the vascular device. These cells were analyzed with respect to messenger RNA and protein expression. By using immunocytochemical and single-cell reverse transcription polymerase chain reaction (PCR), we found that endothelial gene expression can be monitored. The methods we developed allow performance of endothelial biopsy and the opportunity to add a new dimension to our knowledge of vascular biology; namely, endothelial properties at the site of developing lesions. We hence introduce this technology. The purpose of this study was to demonstrate the feasibility of this technology and to explore possible applications.
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MATERIALS AND METHODS |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONAppendix 1References |
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To demonstrate that endothelial samples can be obtained from carotid arteries, four patients with carotid arterial stenosis who were undergoing angioplasty and stent placement were recruited. In addition, three patients with cancer who were undergoing percutaneous indwelling central catheter placement and one patient who was undergoing pulmonary angiography were recruited for the purpose of sampling the superior vena cava and pulmonary arteries, respectively.
General surgical consent, which allowed for performance of laboratory studies of tissues removed during the endovascular procedures, was obtained from each patient. Because the biopsy method involved only in vitro processing of existing guide wires that were used in routine fashion during the endovascular procedures, the institutional review board decided that no additional consent was required.
Biopsy and Isolation of Human Endothelial Cells
To perform biopsy (Fig 1) of iliac endothelium, a coaxial curved stainless steel wire with a 0.038-inch diameter, 3-mm curve radius, and heparin coating (J-wire; Cook, Bloomington, Ind) was inserted into the iliac artery through a Potts needle (Becton Dickinson, Franklin Lakes, NJ), and a femoral sheath (Cook) was advanced over the wire. The wire was then pulled back through the femoral sheath, and the tip of the wire was cut at about 3 inches (7.6 cm) and transferred in sterile fashion to a test tube or Petri dish that contained 25 mL of endothelial cell dissociation buffer (0.5% bovine serum albumin, 2 mmol/L ethylenediaminetetraacetic acid, and 100 µg/mL heparin in calcium- and magnesium-free phosphate- buffered saline solution), which was designed to inhibit adhesion of endothelial cells. After 15 minutes of incubation at room temperature and at least 10 rinses with endothelial dissociation buffer (performed with serologic pipets), cells were centrifuged and resuspended in Red Blood Cell Lysing Buffer (Sigma Chemical, St Louis, Mo) to remove contaminating red blood cells. The nonlysed cells were washed once with endothelial cell dissociation buffer and were resuspended in 50 µL of this buffer. A smear of 5 µL of the cell suspension was stained with a modified Wright stain kit (Leukostat; Fisher, Springfield, NJ). The yield of endothelial cells was estimated by counting cells that showed typical endothelial morphology at light microscopy.
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Culture of Control Endothelial Cells
Human umbilical vein endothelial cells (HUVECs), HUV-EC-C, ATCC CRL-1730, were obtained from the American Type Culture Collection (Rockville, Md). They were grown in either F12K medium (Life Technologies, Rockville, Md), as recommended by American Type Culture Collection, or EB medium (Clonetics, Walkersville, Md). These cells were used to demonstrate typical morphology of endothelial cells and as control cells for evaluation of cell viability and reverse transcription PCR assay results. To activate endothelial cells for reverse transcription PCR analysis of endothelial gene expression, HUVECs and patient-derived endothelial cells were treated with 50 ng/mL lipopolysaccharide at 37°C for 6 hours.
Immunocytochemical Confirmation of Endothelial Cell Type and Detection of Cell Adhesion Molecules
Immunocytochemical analysis is a technique for detection of cellular proteins by using antibodies that specifically recognize these proteins. Antibodies generated in different species can be applied together to localize two proteins at the same time. This technique was used to verify the endothelial identity of patient-derived cells. We double-labeled these cells with combinations of endothelial markers: rabbit anti–human von Willebrand factor (vWF) (Sigma Chemical); goat anti–angiotensin-converting enzyme, produced in the laboratory of one of the authors (D.M.S.); and mouse monoclonal antithrombomodulin (DAKO, Carpinteria, Calif). Antibodies to the leukocyte marker CD45 (also known as common leukocyte antigen) (Sigma Chemical) and to vascular smooth muscle 
-actin (Sigma Chemical) were used as negative control markers.
Immunocytochemical analysis was also used to assess the activation state of patient-derived endothelial cells by using goat anti–human vascular cell adhesion molecule 1 (VCAM-1), goat anti–human intercellular adhesion molecule 1 (ICAM-1), or goat anti–human E-selectin (R&D, Minneapolis, Minn). Endothelial cells were identified on the basis of the presence of vWF.
Slides of isolated cells were prepared by using a cytocentrifuge and were fixed in 4% paraformaldehyde. Because treatment with Triton X-100 did not affect vWF staining but weakened the signal for membrane thrombomodulin, we did not pretreat the cells with Triton X-100. Sites of primary antibody binding were visualized with affinity-purified fluorescein- and rhodamine-labeled donkey antimouse, antigoat, and antirabbit antibodies (Jackson ImmunoResearch, West Grove, Pa).
Assays for Endothelial Cell Viability
We used two independent assays to evaluate the viability of patient-derived endothelial cells. The first assay was used to assess the membrane integrity of endothelial cells (LIVE/DEAD Viability/Cytotoxicity kit; Molecular Probes, Eugene, Ore); this kit contained two dyes: ethidium homodimer 1 and calcein AM. Ethidium homodimer 1 stains nuclear DNA red if the integrity of the cell membrane has been violated. Calcein AM is virtually nonfluorescent, but after passage through a cell membrane, it is converted to intensely fluorescent calcein by means of cellular esterase in the lysosomes (15). Therefore, a live cell will have speckled cytoplasmic staining, and a dead cell will show a collection of red fluorescence in the nucleus.
Patient-derived endothelial cells were resuspended in 200 µL of calcium- and magnesium-free phosphate-buffered saline solution with 0.5% bovine serum albumin and were plated in a well of an eight-well chamber slide (Lab Tek; Fisher) that had been coated with 0.1% gelatin. The cells were incubated with 2 mmol/L ethidium homodimer 1 and 4 mmol/L calcein AM for 
hour before fixation in 4% paraformaldehyde. Endothelial cells were identified by observing their large size, and the numbers of live and dead cells were determined with the aid of a fluorescent microscope. HUVECs were processed in parallel as control cells. One-third of HUVECs were not exposed to dyes and served as negative control cells, whereas another one-third of HUVECs were permeabilized by means of incubation in 0.1% saponin for 10 minutes prior to staining to provide a positive control for dead cells.
The second assay, the incorporation of acetylated low-density lipoprotein labeled with 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), was used to test the metabolic function of endothelial cells. Acetylated low-density lipoprotein is taken up by endothelial cells through their scavenger receptors (16). This process is energy dependent and, thus, occurs only in metabolically active cells. The uptake of fluorescent DiI-labeled acetylated low-density lipoprotein also has been used to help identify endothelial cells (16).
After isolation, the number of patient-derived endothelial cells was determined by using a modified Wright stain. These cells were suspended in 200 µL of F12K medium that contained 10 mmol/L DiI-labeled acetylated low-density lipoprotein (Molecular Probes) and were plated on eight-well chamber slides (Lab Tek; Fisher). Cells were incubated for 4 hours before fixation in 4% paraformaldehyde. A similar number of HUVECs were processed at the same time as control cells. Endothelial cells were identified by observing their large size and were counter stained with anti–angiotensin-converting enzyme antibody.
Single-Cell Reverse Transcription PCR
Reverse transcription PCR is a standard molecular biologic technique that allows detection of tiny amounts of messenger RNA in cells and tissues. Specific segments of messenger RNA molecules are converted to complementary DNA molecules by means of reverse transcriptase and are amplified more than a millionfold by means of PCR so that these segments of complementary DNA can be detected with routine agarose gel electrophoresis.
Single-cell reverse transcription PCR was used to assess E-selectin gene expression in patient-derived cells to demonstrate the feasibility of detecting messenger RNA with this technique. This experiment was performed with four random samples of endothelial cells derived from iliac arteries. In each experiment, 20 individual endothelial cells were identified on the basis of size and shape by using an inverted–phase-contrast microscope. The cells were picked up with a micropipet and transferred to thin-walled PCR tubes that contained a detergent-based lysis buffer. Two rounds of nested PCR were used to examine gene expression in endothelial cells. Three pairs of primers were simultaneously added to the PCR tubes: vWF, which served as an endothelial cell marker (17); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (18), which served as a positive control and internal standard; and E-selectin, which served as an indicator of endothelial activation (19). (See the Appendix for the sequences of the nested primers.) At least one set of the nested primers for each gene is located on a different exon.
A Titan reverse transcription PCR system (Boerhinger, Indianapolis, Ind) was used for reverse transcription PCR by following the manufacturer's instructions. In brief, cell lysis was carried out at 72°C, and reverse transcription was accomplished at 50°C for 30 minutes and at 60°C for 15 minutes. We used 20-µL volumes for 35 cycles of first-round PCR and 25 cycles of second-round PCR, which were performed with a thermocycler (GeneAmp 9600; Perkin-Elmer, Foster City, Calif). The PCR products were analyzed with electrophoresis on 2% agarose gel.
Endothelial Activation and Patient Age
To demonstrate the potential clinical application of endothelial biopsy, we studied the association of endothelial activation with patient age by using endothelial cells obtained from iliac arteries in 10 patients with arteriovenous malformation or aneurysm. The cells were subjected to immunocytochemical analyses with antibodies to E-selectin, ICAM-1, and VCAM-1. The total number of endothelial cells in the specimen was determined by means of double labeling with vWF. The number of endothelial cells that expressed the indicated cell adhesion molecules was determined, and the proportion of positive cells in the samples was calculated. The association of cell adhesion molecules with age was assessed with a linear regression model with logit transformation of the proportion of positive cells as the dependent variable. The effects of sex, diagnosis (aneurysm vs arteriovenous malformation), and hypertension were determined by means of multivariate logistic regression.
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RESULTS |
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Salient features of the cell population dissociated from guide wires introduced into the iliac artery and that were initially identified as endothelial-like were revealed on Wright-stained smears: (a) Cells were more than 20 µm in diameter, which was considerably larger than leukocytes, the principal contaminating cell type; (b) on many occasions, cells were present in small aggregates (two to three cells) or sheets (five or more cells); and (c) individual cells were polygonal with a granular, faint blue cytoplasm, an oval nucleus with fine red-purple chromatin, and, sometimes, a nucleolus (Fig 2).
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-actin), however, deeper layers of the blood vessel wall—namely, smooth muscle cells—were not present in preparations. Thus, it was unlikely that the guide wire penetrated more deeply than the endothelium and some of the contiguous matrix-associated material in the vessel wall. Taken together, the size, shape, and coloration of endothelial cells in these Wright-stained smears allowed us to distinguish them easily from other cells in peripheral blood. Further characterization of cells putatively identified as endothelial was accomplished with antibodies to the endothelial markers angiotensin-converting enzyme (20), thrombomodulin (21), and vWF (22) (Fig 3). Dual-fluorescence microscopy demonstrated the presence of thrombomodulin (Fig 3a) and vWF (Fig 3b) in the same cells. Similarly, a cell positive for angiotensin-converting enzyme (Fig 3c) also was positive for vWF (Fig 3d). Whereas staining for vWF revealed a more punctate appearance, consistent with the presence of vWF in Weibel-Palade bodies, angiotensin-converting enzyme antigen was present more uniformly throughout the cytoplasm, which resulted in a ground-glass appearance, except for the absence of nuclear staining. Although none of these markers are exclusively endothelial (eg, vWF is expressed by both endothelial cells and platelets), only endothelial cells express all three markers.
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-actin were identified (Fig 3g), although the same field had cells that displayed the vWF antigen (Fig 3h). Thus, on the basis of general morphologic criteria and the presence of specific markers, endothelial cells could be distinguished from other nonendothelial contaminants in the cell population dissociated from curved guide wires. Endothelial cells harvested with the same methods from large veins (four patients) had a similar appearance (Fig 2e) and number (mean, 245 cells ± 57). Comparable results were obtained from stenotic carotid arteries (four patients), although the mean number of cells was reduced (25 cells ± six). These data were suggestive of the potential for extrapolation of our method to particular vessels of interest and diverse lesion sites.
Viability of Patient-derived Endothelial Cells
Cell viability was most likely to be compromised during harvesting from the vessel wall and cell processing. Two independent methods, the viability assay (Fig 4b) and uptake of DiI-labeled acetylated low-density lipoprotein (Fig 4d, 4f), were used to evaluate cellular integrity in two random endothelial samples from iliac arteries. As a control for cell recovery, similar numbers of HUVECs were processed in parallel with cells dissociated from the guide wires. Green calcein-AM staining of live cells versus uptake of red ethidium homodimer 1 by dead cells was observed (Fig 4b). Uptake of DiI-labeled acetylated low-density lipoprotein in HUVECs (Fig 4d) resulted in a speckled appearance of the dye in a cellular distribution. Cells from the guide wires also displayed uptake of DiI-labeled acetylated low-density lipoprotein (Fig 4f).
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Reverse Transcription PCR Analysis of Transcripts in Endothelial Cells
The small final number of cells available for further study from patient samples (50 cells per sample, two patients) necessitated the use of sensitive molecular biologic techniques for analysis of cellular properties. For this reason, we developed a single-cell reverse transcription PCR method for simultaneous assessment of the expression of many genes in each cell.
After the cell isolation procedure, 20 cells with endothelial features were randomly selected from each sample for single-cell reverse transcription PCR by using primers for GAPDH and vWF (Fig 5). Approximately 20% of the cells (from five samples) yielded amplicons for GAPDH and vWF (Fig 5). This was consistent with the 25% viability of recovered endothelial cells as estimated with the viability and DiI-labeled acetylated low-density lipoprotein uptake assays. Control experiments with freshly prepared HUVECs, in which virtually all cells were viable, showed 100% of cells expressing transcripts for vWF (Fig 5).
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Expression of Leukocyte Adhesion Molecules in Endothelial Cells
Endothelial cell expression of cell adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1, is altered with vascular perturbation (25). E-selectin has been most closely linked to acute inflammation and recruitment of leukocytes in postcapillary venules (26). Changes in ICAM-1 and VCAM-1 often are more sustained and associated with chronic conditions such as atherosclerosis (27). For example, VCAM-1 has been detected at the earliest stages of lesion development in genetically manipulated murine models of atherosclerosis (28), and increased levels of a soluble form of VCAM-1 have been noted (27,28) in cases of diabetes and other conditions associated with a high risk of vascular disease.
These considerations led us to analyze expression of these adhesion molecules in iliac arteries in 10 consecutive patients, four with a cerebral arterial aneurysm and six with arteriovenous malformation in the brain. The seven female and three male patients, four of whom were hypertensive, were 15–67 years of age. Figure 6 shows representative results from cells isolated from one patient. Immunostaining of patient-derived cells initially adherent to curved guide wires showed expression of E-selectin (Fig 6a) in a population of cells distinct from those with vWF (Fig 6b, 6c). Absence of E-selectin in endothelial cells harvested from patients was not due to methodological problems in cell isolation or to the detection procedure, because identically processed HUVECs treated with lipopolysaccharide showed strong E-selectin expression (data not shown).
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DISCUSSION |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONAppendix 1References |
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These data emphasize the ease with which endothelial cells are dislodged during vascular procedures and are in keeping with previous electron microscopic studies (29) and observations of circulating endothelial cells after endovascular procedures (30,31). Even without the use of instrumentation, endothelial cells can become detached and circulate in blood (32). Especially in sickle cell disease, the authors of careful studies (32) have documented isolation of endothelial cells by using a monoclonal antibody and found that the number of circulating endothelial cells increases during crises. Thus, release of endothelial cells into the blood is likely to occur in many conditions and at an exaggerated rate during vascular perturbation.
Circulating and/or easily detached endothelial cells may also be considered a physiologic occurrence in two settings. Dividing endothelial cells display diminished adherence to the underlying substrate and, thus, are more likely to detach. Chen et al (33) showed an increase in the number of dividing cells at arterial bifurcations, which are sites of turbulent flow; this correlates with an increase in vascular permeability at these sites, which is consistent with a looser attachment of dividing endothelial cells to neighboring cells in the monolayer. In a pathologic context, tumor angiogenesis and other vascular responses that result in new vessel formation and/or remodeling also result in an increase in endothelial turnover and a looser association of endothelium with vessel wall (34,35).
Alternatively, circulating endothelial cells may represent endothelial progenitor cells ready to be incorporated into remodeling peripheral endothelium. Asahara and colleagues (36) have identified a population of circulating endothelial progenitor cells on the basis of the expression of CD34, vascular endothelial growth factor receptor 2, endothelial nitric oxide synthase, CD31, and tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor–like domains 2 (referred to as Tie-2).
In contrast to the process of angiogenesis, in which vessels were formed exclusively as an outgrowth of previously formed vasculature, the results reported by Asahara et al (36) have emphasized a contribution of vasculogenesis in new vessel formation. In vasculogenesis, circulating endothelial progenitor cells become adherent to the vessel wall and especially populate sites of ongoing new vessel formation. The source of endothelial progenitor cells is unknown at present, although the common expression of CD34 and vascular endothelial growth factor receptor 2 by endothelial progenitor cells and hematopoietic stem cells suggests that they may derive from bone marrow. The ease we experienced in detaching endothelial cells from peripheral vessels raises the possibility that endothelial progenitor cells may derive from peripheral vessels, especially in the presence of angiogenic stimuli.
The extraordinarily high yield of endothelial cells from a patient with a highly vascular meningioma may have been an incidental finding or may reflect pathologic changes in the endothelium. Under the influence of tumor angiogenesis, there may be increased proliferation in remote vessels, as well. Alternatively, more endothelial progenitor cells may be generated from peripheral vessels or incorporated in those vessels. The possibility of a relationship between angiogenesis and yield of endothelial cells appears to be an appropriate subject for further study.
After a typical endovascular procedure, about 250 cells dissociated from a curved guide wire displayed endothelial-like features on the basis of evaluation of Wright-stained smears. After our centrifugation and adherence procedure, which required 75 minutes, approximately 50 endothelial cells were available for further study. The morphology, viability, and reactivity of these cells to lipopolysaccharide suggested that these cells were random samples of the endothelium rather than sick cells that were ready to fall off. However, we cannot exclude the possibility that less adherent cells, such as dividing cells, were preferentially sampled. Because of the small number of cells obtained, we emphasized single-cell approaches to the analysis of patient-derived endothelium. Although it was a challenge, we showed that methods for analysis of single cells at the protein and messenger RNA levels are available and can be applied with our samples.
Because simultaneous visualization of up to three markers on the same cell is possible with the aid of immunocytochemical techniques, analysis with an accepted endothelial marker, such as vWF, and with two activation markers of interest certainly is feasible. This method would allow definitive characterization of the cell under study as endothelial in origin and would permit detection of additional antigens. Such other markers could include endothelial receptors for growth factors, developmental regulators and/or regulators of the blood coagulation mechanism, the presence of the TAT protein (37), or Bartonella-associated antigens (38) in patients suspected of having syndromes related to infection with the human immunodeficiency virus.
The most exciting implication of our results is the isolation of intact messenger RNA from patient-derived endothelial cells. By using single-cell reverse transcription PCR, transcripts in endothelial cells can be amplified, and, in the same sample, the presence of known markers can be detected. Furthermore, complementary DNA generated from the small amounts of messenger RNA in the cell samples provides the basis for construction of complementary DNA libraries to be used in the analysis of unexpected and unknown molecular species expressed by cells of the vessel wall. Thus, in the same cell, its endothelial origin can be ascertained by observing the presence of transcripts for vWF and potential activation markers, as well as previously unrecognized messenger RNA species, and these can be determined simultaneously.
There are caveats with respect to the use of reverse transcription PCR in such differential display strategies. For example, gene expression at the single-cell level may be variable, and selection bias must be carefully avoided. Our method will allow selection of multiple cells from any sample to serve as a starting point for comparisons with cells from the same individual harvested from a different location or with cells harvested from another patient.
In summary, our data demonstrate that viable endothelial cells suitable for molecular analysis at the transcript and protein levels can be obtained during routine angiographic procedures. Our initial results, which are based on a small sample, showed a trend toward an increase in VCAM-1 expression with increased age and the absence of the activation marker E-selectin in the endothelial cell population. Although further studies are needed to help strengthen these conclusions, patient-derived endothelial cells provide a useful tool for future analysis of vascular disease by using immunocytochemical and single-cell reverse transcription PCR methods.
Potential insights from the application of this technique to the comparison of endothelial cell gene expression from lesional or lesion-prone areas of the vessel wall in quiescent cells could be far-reaching. The same approach may also be applied to animal models of vascular diseases or be used to sample endothelial cells from various vascular segments in experimental animals such as nonhuman primates. The molecular techniques used in this study are standard and mature techniques that are routinely performed by molecular biologists, as well as by investigators in clinical laboratories.
Insights gained from the results of these studies offer promise for the stratification of disease, monitoring of therapeutic effects, and guidance for therapeutic interventions, including provision of targets for gene therapy. Nonetheless, it is essential to state that, as of yet, we have not obtained satisfactory numbers of endothelial cells from stenotic carotid arteries (an example of a vascular lesion) to allow molecular analysis. Modification of our procedure and the type of device used may be necessary at such sites, where high-velocity blood flow is in the opposite direction of wire retrieval. The results of the current study strongly suggest that efforts to pursue such an enhancement of the technique are warranted.
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Appendix 1 |
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TOPAbstractIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONAppendix 1References |
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The sequences for the GAPDH primers were 5'-GGACCTGACCTGCCGTCTAGA- 3' (730–750), 5'-GGTACATGACAAGGTGCGGCT-3' (1,183–1,163), 5'-GGTGGTGAAGCAGGCGTCGGA-3' (781–801), and 5'-GGTCTACATGGCAACTGTGAG-3' (1,137–1,117).
The sequences for the E-selectin primers were 5'-CCTCTGACAGAAGAAGCCAAG-3' (381–401), 5'-CTGCAAACCAGGCTTCCATGC-3' (697–677), 5'-CAG GTGAACCCAACAATAGGC-3' (412–432), and 5'-CACTTGAGTCCACTGAAGCCA-3' (637–617).
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Acknowledgments |
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Footnotes |
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Abbreviations: DiI = 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate GAPDH = glyceraldehyde-3- phosphate dehydrogenase HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule 1 PCR = polymerase chain reaction VCAM-1 = vascular cell adhesion molecule 1 vWF = von Willebrand factor
Author contributions: Guarantors of integrity of entire study, L.F., D.M.S., J.P.S.; study concepts, L.F., J.P.S.; study design, L.F., D.M.S.; definition of intellectual content, L.F., D.M.S., J.P.S.; literature research, L.F.; clinical studies, L.F., J.P.S.; experimental studies, L.F.; data acquisition, L.F.; data analysis, L.F., D.M.S.; statistical analysis, L.F., J.P.S.; manuscript preparation and editing, L.F., D.M.S.; manuscript review, J.P.S.
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