Address of Corresponding Author

J Vasc Res 2005;42:174-180 (DOI: 10.1159/000084406)

 Outline

• Key Words
• Abstract
• Introduction
• Methods
• Collection and Labelling of PBMC
• PBMC Injection and Silastic Tube Procedures
• Immunofluorescence and Immunohistochemistry
• Histomorphometry and Quantitative Analysis
• Statistical Analysis
• Results
• Discussion
• Acknowledgments
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Myofibroblasts
• Macrophages
• Transdifferentiation
• Stem cells

  Abstract

Background: Bone marrow-derived cell populations possess progenitor cell capacities. Emerging evidence also suggests significant plasticity of differentiated mononuclear cell lineages. We therefore assessed the distribution of transplanted peripheral blood mononuclear cells (PBMCs) in granulation tissue formation, and evaluated their possible transdifferentiation into myofibroblasts. Methods: Silastic tubes were inserted into the peritoneal cavity of rats, followed by injection of PKH26-labelled PBMCs isolated from donor animals. At 3, 14 and 21 days, the distribution of PKH26⁺ cells as well as their colocalization with myofibroblast/smooth muscle cell [-smooth muscle (-SM) actin] or macrophage markers (ED1/ED2) were determined. Results: Round-shaped PKH26⁺ cells accumulated around the implants at 3 days, while myofibroblasts were rare. Later, peritoneal granulation tissue constituted an inner, multilayered capsule primarily comprising -SM actin⁺ cells that was surrounded by more loosely organized inflammatory connective tissue. PKH26-labelled, spindle-shaped cells were abundantly found in tissue capsules. As a key finding, granulation tissue at 14 and 21 days contained cells with both PKH26 and -SM actin labelling. Accordingly, a subpopulation of cells staining positive for macrophage markers showed a spindle-shaped morphology and -SM actin expression. Conclusions: Transplanted PBMCs contribute to granulation tissue, and acquire myofibroblast characteristics during de novo tissue formation. Mononuclear cells may transdifferentiate into myofibroblast-like cells within an inflammatory environment.

Copyright © 2005 S. Karger AG, Basel

 Introduction

Concepts of cell lineage and differentiation are currently enlarged for pluripotent stem cells and circulating progenitor cells with wide-ranging transdifferentiation capacities. Although several cell populations with progenitor-like properties have been characterized in cardiovascular pathophysiology, their exact ontogeny and mechanisms of their recruitment and homing are incompletely understood. Settings of progenitor cell activity are often inflammatory sites, and in vitro propagated progenitor cell populations frequently originate from peripheral blood mononuclear cell (PBMC) preparations [1, 2, 3, 4]. Hence, the presence of an inflammatory environment and/or inflammatory cells may be linked to progenitor cell-attributed effects.

Inflammation-associated de novo tissue formation had been reported to occur around biocompatible Silastic tubes inserted into the peritoneal cavity of different species [5, 6]. These granulation tissue tubes resembled blood vessels both in transmural architecture and protein expression patterns, and indeed constituted functional vascular grafts [5]. In this model, bone marrow transplantation experiments revealed marrow-derived cells to contribute to tissue formation, possibly by transdifferentiation into smooth muscle cells (SMCs) or myofibroblasts [6, 7]. As to inflammation-associated tissue formation, earlier studies had shown that cultured macrophages from buffy coat or peritoneal exudate gain connective tissue-forming capacity [8, 9]. Recently, a CD34⁺ PBMC subpopulation was found to transdifferentiate into SMCs, endothelial and cardiac muscle cells, a process significantly augmented by local tissue injury like experimental myocardial infarction [10]. Also, cultured SMCs can acquire macrophage-related gene expression upon cholesterol loading, suggesting a transdifferentiation cross-link between both cell types [11].

Having the perspective to determine whether also peripheral, primarily circulating mononuclear cells can contribute to inflammation-associated tissue generation, we assessed the distribution of ex vivo PKH26-labelled PBMCs in rat peritoneal granulation tissue. To characterize properties and specific distribution of participating cell types, we determined the presence and distribution of macrophage markers at different stages of inflammatory tissue organization, as well as the expression pattern of -smooth muscle (-SM) actin identifying myofibroblasts or SMCs. Since a proportion of transplanted PBMCs apparently acquired myofibroblast characteristics, our findings (1) show that the origin of some tissue-organizing cells in this model is from peripheral blood, (2) underscore PBMC plasticity, and (3) suggest a myofibroblastoid transdifferentiation capacity of these cells.

 Methods

 Collection and Labelling of PBMC

Male Sprague-Dawley rats (350-450 g) were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (5 mg/kg). After discarding the first 300 µl, whole blood was harvested from carotid arteries of 2 donor rats per recipient animal. PBMC were isolated by density gradient centrifugation using Histopaque-1077 (Sigma, St. Louis, Mo., USA). After removal of platelets and red blood cells, PBMCs were stained with PKH26-GL Red Fluorescent Cell Linker Kit (Sigma), a fluorescent dye previously used for in vitro proliferation studies and in vivo cell tracking [12, 13, 14]. Cell viability as determined by trypan blue and propidium iodine exclusion was >95% after the staining procedure, and flow cytometry showed PKH26 labelling of >98% of the cells.

 PBMC Injection and Silastic Tube Procedures

Sprague-Dawley rats were anesthetized as described above. Following laparotomy and surgical preparations, a total of approximately 9 × 10⁷ PKH26-labelled PBMCs were injected into the inferior vena cava. Thereafter, 3 sterile Silastic silicone tubes (Baxter, Deerfield, Ill., USA) with outer diameter of 2 mm and length of 10 mm were inserted into the peritoneal cavity. Tubes surrounded by tissue capsules were harvested after 3 (n = 3), 14 (n = 9) and 21 days (n = 5), and tubing was separated from tissue. Liver, lung, skin and spleen were also excised, and all specimens were fixed in paraformaldehyde. All animal studies were approved by the Emory University Institutional Animal Care and Use Committee.

 Immunofluorescence and Immunohistochemistry

Monoclonal murine -SM actin (1:500; clone 1A4; Sigma), CD68 (1:200; clone ED1; Serotec, Oxford, UK), and CD163 (1:500 or 1:1,000; clone ED2; Serotec) antibodies were used.

In brief, for immunofluorescence staining, frozen tissue sections were thawed, fixed in acetone, dried, and rehydrated in PBS. All primary antibodies were applied at the indicated dilutions in 1.0% BSA in PBS. Sections were washed in PBS and incubated with a biotinylated secondary antibody (horse anti-mouse IgG; 1:400; Vector Laboratories, Burlingame, Calif., USA) in PBS containing 1.0% BSA and 2.0% normal horse serum. Following an additional washing step, fluorescein-streptavidin (1:100; Vector) was applied. Nuclei were counterstained with a 0.25 µg/ml DAPI solution (Sigma).

Immunohistochemical double staining was done as previously described [15] by incubating first with primary anti--SM actin and secondary biotinylated horse anti-mouse IgG antibodies (1:400; Vector). ABC-alkaline phosphatase (Vector) was applied with Vector Red as chromogenic substrate. Slides were incubated with biotin solution (Vector), dehydrated in graded alcohol, and treated with Histo-Clear (National Diagnostics, Atlanta, Ga., USA). Endogenous peroxidase was blocked with 0.3% H₂O₂/methanol, and slides were incubated with the second primary ED1/ED2 antibody, respectively. Biotinylated horse anti-mouse IgG (1:400; Vector) was applied, and sections were stained with the ABC-peroxidase kit (Vector) and diaminobenzidine, yielding a brown reaction product. Nuclei were counterstained with hematoxylin. Similar results were observed with single-antibody staining in every case. Serial sections treated with secondary antibodiesonly or with nonimmune IgG did not show any staining.

 Histomorphometry and Quantitative Analysis

The density of PKH26⁺-SM actin⁺ and PKH26⁺-SM actin^- cells was evaluated within the inner tissue capsule at 14 and 21 days. PKH26-labelled cell density was also determined for liver, lung, skin and spleen sections, where double labelling with -SM actin was not observed. High-resolution digital photomicrographs were taken with an RT Slider video camera (Diagnostic Instruments, Sterling Heights, Mich., USA) and the Spot software (version 3.5.6 for MacOS, Diagnostic Instruments), and 8 randomly selected areas (0.15 ± 0.04 mm²) per tissue cross section were analyzed by 2 independent investigators. No significant interobserver variations were noted.

 Statistical Analysis

Data are presented as mean number of labelled cells per mm² ± SD. Statistical analysis was done with the two-tailed unpaired Student t test for comparisons between the means of two groups. Values of p < 0.05 were considered significant.

 Results

Silastic tubes were harvested from the peritoneal cavity at 3, 14 and 21 days after implantation. Regions adjacent to the foreign material contained accumulations of round-shaped cells at day 3, followed by the formation of several fibrous connective tissue layers in areas directly contacting a tube at later time points (fig. 1, 2). At days 14 and 21, these innermost regions constituted a multilayered capsule surrounded by more loosely organized tissue with microvessels and aggregations of round-shaped cells (fig. 2, 3, 4). A discontinuous layer of oval mesothelial cells demarcated these capsules from the peritoneal cavity.

Fig. 1. Immunofluorescence staining of inflammatory tissue surrounding abdominal tubes at day 3; digitally merged images. A Photomicrograph of early granulation tissue formation showing accumulation of donor-derived, PKH26-labelled PBMCs (red; arrowheads). Microvascular wall exhibiting -SM actin immunofluorescence (green; arrow). No yellow merging color indicative of PKH26/-SM actin colocalization is detected. B Immunofluorescence staining for the macrophage marker ED2 in loosely organized granulation tissue. Host-derived macrophages (green; thin arrows) can be distinguished from PKH26-labelled, transplanted cells that also bear ED2 (yellow merging color; arrows), while only very few, small PKH26+ cells do not show macrophage marker expression (red; arrowhead). * = Silastic tube area. DAPI nuclear counterstaining (blue). Bar = 30 µm.

Fig. 2.-SM actin immunofluorescence (A, D) and PKH26 staining (B, E) in peritoneal granulation tissue at day 14 (A-C) and 21 (D-F); digitally merged photomicrographs in C and F. The dense inner capsule mainly consists of -SM actin+ cells (green). While PKH26-labelled cells without -SM actin expression (red; arrowheads) are predominantly located in peripheral tissue layers, some donor-derived, PKH26+ former PBMCs cells express -SM actin within the inner capsule (yellow merging color; arrows; C and F). The inlay in C shows a high-power magnification of the box area with a spindle-shaped PKH26+-SM actin+ cell (yellow) and a round-shaped PKH26+-SM actin- cell (red). * = Silastic tube area. DAPI nuclear counterstaining (blue). Bar = 30 µm.

Fig. 3. Immunofluorescence photomicrograph of the granulation tissue capsule at day 14, demonstrating the spindle-shaped morphology of ED1+ macrophages of both donor (yellow merging color; arrows) and host origin (green; small arrows). * = Silastic tube area. DAPI nuclear counterstaining (blue). Bar = 30 µm.

Fig. 4. High-magnification photomicrographs of double immunostaining in granulation tissue sections at day 14 (A) and 21 (B). In addition to mono-labelled ED2+ granulation tissue macrophages (brown; small arrows) and -SM actin+ myofibroblasts/SMCs (red; arrowheads), some spindle-shaped cells reveal coexpression of the macrophage marker ED2 (brown) and -SM actin (red) in the granulation tissue capsule (arrows). Bar = 30 µm.

Tissue surrounding the Silastic material at day 3 frequently contained PKH26-labelled cells originating from donor PBMC preparations, while -SM actin⁺ cells were scarce (fig. 1A). To determine PKH26⁺ cell types within this de novo forming granulation tissue, and to distinguish donor- from host-derived macrophages, immunostaining for the rat macrophage markers ED1 and ED2 was done. Digital merging of macrophage immunofluorescence images with PKH26 photomicrographs showed - besides host-derived ED1⁺/ED2⁺ cells - numerous donor-derived PKH26⁺ cells with macrophage marker expression, indicating a mix of donor- and host-derived macrophages in early granulation tissue (fig. 1B). With ongoing tissue organization at days 14 and 21, -SM actin⁺ cells formed the multilayered inner capsule around the implant, while -SM actin immunofluorescence in more peripheral regions was mostly confined to microvessels (fig. 2). As a key finding, tissue capsules at 14 and 21 days contained donor-derived PKH26⁺ cells staining positive for -SM actin (402 ± 223 and 333 ± 167 PKH26⁺-SM actin^- cells/mm², 59 ± 21 and 47 ± 17 PKH26⁺-SM actin⁺ cells/mm² at 14 and 28 days, respectively; p = nonsignificant; fig. 2). Furthermore, donor-derived PKH26⁺ cells within the inner, dense compartment revealed a spindle-shaped morphology at these later time points of tissue organization, contrasting round- shaped PKH26⁺ cells in more loosely organized outer layers (fig. 2, 3). To prove that cells of the monocyte/macrophage lineage are indeed expressing -SM actin in this model, additional double immunostaining experiments were performed. These showed that coexpression of both determinants indeed occurred in some spindle-shaped cells within the inner capsule (fig. 4).

As to the systemic distribution of transplanted PBMCs, sections from different organs were examined for the presence of PKH26-labelled cells. At all time points examined, accumulation of PKH26⁺ cells was observed in spleen sections, where they densely accumulated in the marginal zone around lymphoid follicles and in the red pulp (5,530 ± 1,675 PKH26⁺ cells/mm²; fig. 5A). Hepatic tissue revealed a more scattered pattern (2,422 ± 1,013 PKH26⁺ cells/mm²; fig. 5B), and few if any PKH26⁺ cells were detectable in lung and skin sections (not shown).

Fig. 5.Systemic distribution of transplanted PKH26-labelled PBMCs. A Spleen section showing a secondary lymphoid follicle with dense circumferential aggregation of PKH26+ cells. B Periportal area in a liver section. Note spread distribution pattern of transplanted PBMCs throughout the hepatic tissue. DAPI nuclear counterstaining (blue). Bar = 30 µm.

 Discussion

In the present study, we assessed the distribution of ex vivo PKH26-labelled PBMCs in a rat model of peritoneal granulation tissue formation around biocompatible Silastic tubes. Numerous PKH26⁺ cells were detected within these tissue capsules, mostly characterized as macrophages (fig. 1, 2). As a central finding, a proportion of PKH26-labelled PBMCs revealed -SM actin immunolabelling and spindle-shaped morphology by day 14 and 21 (fig. 2, 3, 4). These findings point to a direct contribution of primarily circulating PBMCs to inflammation-associated tissue organization, and suggest their transdifferentiation into myofibroblasts or SMC-like cells.

Thorough studies in a similar animal model had previously identified bone marrow-derived cells to participate in de novo tissue formation, suggesting a systemic origin of -SM actin⁺ myofibroblast progenitor cells [6]. However, bone marrow preparations may not only contain erythroblastic and myeloblastic progenitors, but also stromal cells and mature blood cells. To the best of our knowledge, this is the first study to show that peripherally circulating PBMCs also possess the capacity to acquire myofibroblast characteristics in vivo, thereby gaining tissue-organizing capacity. The absence of PKH26⁺-SM actin⁺ cells at day 3 - despite the abundance of PKH26⁺ cells - supports the concept that primarily circulating PBMCs transdifferentiate into myofibroblasts during later phases of tissue organization in an inflammatory environment (fig. 1). Indeed, earlier work had shown that monocytes can acquire a SMC-like phenotype and synthesize extracellular matrix components in vitro [8, 9]. Also, cells with intermediate characteristics between macrophages and SMCs were detected in human aortal sections as well as in neointimal lesions post-porcine coronary angioplasty [16, 17]. A transdifferentiation link between SMCs and macrophages is further supported by a recent report showing SMC transdifferentiation into a macrophage-like phenotype [11]. It has also been reported that treatment of murine macrophage cell lines with the inflammatory cytokine interferon- can induce -actin expression and an SMC phenotype in these cells [6].

Recipient animals in our study were not depleted from their own PBMCs, and macrophage markers were abundantly found on spindle-shaped cells within the inner capsule, colocalized with -SM actin expression (fig. 2, 3, 4). Therefore, the incidence of PBMC-myofibroblast transdifferentiation in this model may be even larger, besides generally accepted mechanisms of granulation tissue formation like the migration of local fibroblasts with subsequent myofibroblast differentiation [18]. Also, we cannot exclude that some labelling of the injected cells was diluted in proliferating cells, thus further decreasing the number of detectable transdifferentiation. Therefore, the quantification of PKH26⁺ and PKH26⁺-SM actin⁺ cells by fluorescence techniques can only give a rough estimate of the numerical relations. However, the aim of our study was to determine the general principle that primarily circulating PBMCs participate in inflammation-associated tissue formation, and an exact quantification of transdifferentiation will require further investigation. Given the low numbers of circulating progenitor cells in whole blood in vivo [19], PBMCs could be of considerable importance for transdifferentiation effects primarily attributed to progenitors. A wide-ranging transdifferentiation capacity of PBMCs is underlined by a recent report showing that a peripheral blood monocyte-derived subpopulation can differentiate into cell types like endothelial cells, neuronal cells or hepatocytes [20]. Also, a cell population commonly referred to as endothelial progenitor cells obviously possessed monocytic characteristics [21], and not only endothelial progenitor cells but also spleen-derived mononuclear cells are capable of accelerating the repair of endothelial cell damage in adult blood vessels [14].

Our data also reveal that the absolute level of transplanted PBMCs homing to the site of granulation tissue formation is rather low, whereas the majority of PKH26-labelled cells was found in the spleen, and also in the liver. This is in accordance with findings by Aicher et al. [22], who reported that the vast majority of transplanted mononuclear cell-derived endothelial progenitor cells was detected by radioactive labelling in the spleen and to a lesser extend in the liver of rats, whereas only a small percentage showed homing to myocardial infarction areas.

Since Ficoll-gradient PBMC preparations may carry some non-PBMCs, we cannot exclude PKH26 labelling of small amounts of other cell types from donor animals. Also, studies aiming to determine transdifferentiation processes of primarily circulating progenitor cells during tissue remodelling may be hampered by their fusion with local cells [23, 24]. Early work by Ross et al. [25] had demonstrated that wound fibroblasts in parabiosis experiments did not originate from hematogenous precursors. However, by use of carotid blood collection and removal of the most likely vascular wall cell-contaminated initial blood volume collected after catheterization, PKH26 labelling of non-PBMCs was minimized. Also, a lack of PKH26/-SM actin colocalization at day 3 as well as ED1/ED2 expression on the vast majority of PKH26-labelled cells (fig. 1B, 3) at all time points clearly show that predominantly PBMCs were initially labelled, making them the most likely candidate for acquiring myofibroblast characteristics at later time points.

Taken together, a proportion of transplanted, primarily circulating PKH26-labelled PBMCs apparently acquired myofibroblast characteristics in peritoneal granulation tissue, suggesting their transdifferentiation within this inflammatory environment. Our findings underscore PBMC plasticity, and suggest a myofibroblastoid transdifferentiation capacity of these cells.

 Acknowledgments

This work was supported by NIH HL57908 (J.N.W.) and a German Cardiac Society postdoctoral scholarship (A.J.). The authors would like to thank Giji Joseph for her expert technical assistance.

  References

1.

Rafii S, Lyden D: Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003;9:702-712.

2.

Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C, Uchida S, Masuo O, Iwaguro H, Ma H, Hanley A, Silver M, Kearney M, Losordo DW, Isner JM, Asahara T: Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461-468.

3.

Urbich C, Dimmeler S: Endothelial progenitor cells: Characterization and role in vascular biology. Circ Res 2004;95:343-353.

4.

Mathur A, Martin JF: Stem cells and repair of the heart. Lancet 2004;364:183-192.

5.

Campbell JH, Efendy JL, Campbell GR: Novel vascular graft grown within recipient's own peritoneal cavity. Circ Res 1999;85:1173-1178.

6.

Campbell JH, Efendy JL, Han C, Girjes AA, Campbell GR: Haemopoietic origin of myofibroblasts formed in the peritoneal cavity in response to a foreign body. J Vasc Res 2000;37:364-371.

7.

Han CI, Campbell GR, Campbell JH: Circulating bone marrow cells can contribute to neointimal formation. J Vasc Res 2001;38:113-119.

8.

Allgoewer M, Hulliger L: Origin of fibroblasts from mononuclear blood cells: A study on in vitro formation of the collagen precursor, hydroxyproline, in buffy coat cultures. Surgery 1960;47:603-610.

9.

Sappino AP, Schurch W, Gabbiani G: Differentiation repertoire of fibroblastic cells: Expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest 1990;63:144-161.

10.

Yeh ET, Zhang S, Wu HD, Korbling M, Willerson JT, Estrov Z: Transdifferentiation of human peripheral blood CD34+-enriched cell population into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. Circulation 2003;108:2070-2073.

11.

Rong JX, Shapiro M, Trogan E, Fisher EA: Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading. Proc Natl Acad Sci USA 2003;100:13531-13536.

12.

Parish CR: Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 1999;77:499-508.

13.

De Leon H, Ollerenshaw JD, Griendling KK, Wilcox JN: Adventitial cells do not contribute to neointimal mass after balloon angioplasty of the rat common carotid artery. Circulation 2001;104:1591-1593.

14.

Werner N, Junk S, Laufs U, Link A, Walenta K, Boehm M, Nickenig G: Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res 2003;93:e17-e24.

15.

Scott NA, Cipolla GD, Ross CE, Dunn B, Martin FH, Simonet L, Wilcox JN: Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation 1996;93:2178-2187.

16.

Andreeva ER, Pugach IM, Orekhov AN: Subendothelial smooth muscle cells of human aorta express macrophage antigen in situ and in vitro. Atherosclerosis 1997;135:19-27.

17.

Bayes-Genis A, Campbell JH, Carlson PJ, Holmes DR Jr, Schwartz RS: Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair. Atherosclerosis 2002;163:89-98.

18.

Werner S, Grose R: Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835-870.

19.

Hristov M, Erl W, Weber PC: Endothelial progenitor cells: Mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol 2003;23:1185-1189.

20.

Zhao Y, Glesne D, Huberman E: A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA 2003;100:2426-2431.

21.

Rehman J, Li J, Orschell CM, March KL: Peripheral blood 'endothelial progenitor cells' are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 2003;107:1164-1169.

22.

Aicher A, Brenner W, Zuhayra M, Badorff C, Massoudi S, Assmus B, Eckey T, Henze E, Zeiher AM, Dimmeler S: Assessment of the tissue distribution of transplanted human endothelial progenitor cells by radioactive labeling. Circulation 2003;107:2134-2139.

23.

Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW: Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542-545.

24.

Hoofnagle MH, Wamhoff BR, Owens GK: Lost in transdifferentiation. J Clin Invest 2004;113:1249-1251.

25.

Ross R, Everett NB, Tyler R: Wound healing and collagen formation. VI. The origin of the wound fibroblast studied in parabiosis. J Cell Biol 1970;44:645-654.

  Author Contacts

Dr. Josiah N. Wilcox
Medtronic Vascular
3576 Unocal Place, SS-48, 2D0603
Santa Rosa, CA 95403 (USA)
Tel. +1 707 591 2256, Fax +1 707 566 1408, E-Mail Cy.Wilcox@medtronic.com

  Article Information

Received: August 30, 2004
Accepted after revision: January 6, 2005
Published online: March 14, 2005
Number of Print Pages : 7
Number of Figures : 5, Number of Tables : 0, Number of References : 25

  Publication Details

Journal of Vascular Research (Incorporating International Journal of Microcirculation)

Vol. 42, No. 2, Year 2005 (Cover Date: March-April 2005)

Journal Editor: U. Pohl, Munich; G.A. Meininger, College Station, Tex.
ISSN: 1018-1172 (print), 1423-0135 (Online)

For additional information: http://www.karger.com/jvr

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.