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EXPERIMENTAL STUDY

Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia

Shmuel Fuchs, MD^*, Richard Baffour, PhD^*, Yi Fu Zhou, MD^*, Matie Shou, MD^*, Anthony Pierre, BSc^*, Fermin O. Tio, MD, Neil J. Weissman, MD^*, Martin B. Leon, MD, Stephen E. Epstein, MD^* and Ran Kornowski, MD^*

^* the Cardiovascular Research Institute, Washington, DC, USA
Biomedical Research Foundation of South Texas Inc., San-Antonio, Texas, USA
Cardiovascular Research Foundation, Lenox Hill Hospital, New York, New York, USA

Manuscript received December 12, 2000; revised manuscript received January 9, 2001, accepted January 24, 2001.

Reprint requests and correspondence: Dr. Shmuel Fuchs, Cardiovascular Research Institute, Washington Hospital Center, 110 Irving St. Northwest, 4B-1, Washington, DC 20010
sxf6{at}mhg.edu

	Abstract

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OBJECTIVES

We tested the hypothesis that intramyocardial injection of autologous bone marrow (ABM) promotes collateral development in ischemic porcine myocardium. We also defined, in vitro, whether bone marrow (BM) cells secrete vascular endothelial growth factor (VEGF) and macrophage chemoattractant protein-1 (MCP-1).

BACKGROUND

The natural processes leading to collateral development are extremely complex, requiring multiple growth factors interacting in concert and in sequence. Because optimal angiogenesis may, therefore, require multiple angiogenic factors, we thought that injection of BM, which contains cells that secrete numerous angiogenic factors, might provide optimal therapeutic angiogenesis.

METHODS

Bone marrow was cultured four weeks in vitro. Conditioned medium was assayed for VEGF and MCP-1 and was added to cultured pig aortic endothelial cells (PAEC) to assess proliferation. Four weeks after left circumflex ameroid implantation, freshly aspirated ABM (n = 7) or heparinized saline (n = 7) was injected transendocardially into the ischemic zone (0.2 ml/injection at 12 sites). Echocardiography to assess myocardial thickening and microspheres to assess perfusion were performed at rest and during stress.

RESULTS

Vascular endothelial growth factor and MCP-1 concentrations increased in a time-related manner. The conditioned medium enhanced, in a dose-related manner, PAEC proliferation. Collateral flow (ischemic/normal zone x 100) improved in ABM-treated pigs (ABM: 98 ± 14 vs. 83 ± 12 at rest, p = 0.001; 89 ± 18 vs. 78 ± 12 during adenosine, p = 0.025; controls: 92 ± 10 vs. 89 ± 9 at rest, p = 0.49; 78 ± 11 vs. 77 ± 5 during adenosine, p = 0.75). Similarly, contractility increased in ABM-treated pigs (ABM: 83 ± 21 vs. 60 ± 32 at rest, p = 0.04; 91 ± 44 vs. 36 ± 43 during pacing, p = 0.056; controls: 69 ± 48 vs. 64 ± 46 at rest, p = 0.74; 65 ± 56 vs. 37 ± 56 during pacing, p = 0.23).

CONCLUSIONS

Bone marrow cells secrete angiogenic factors that induce endothelial cell proliferation and, when injected transendocardially, augment collateral perfusion and myocardial function in ischemic myocardium.

Abbreviations and Acronyms   ABM = autologous bone marrow   BM = bone marrow   H&E = hematoxylin and eosim   LTCM = long-term culture medium   MCP-1 = macrophage chemoattractant protein-1   PAEC = pig aortic endothelial cells   VEGF = vascular endothelial growth factor

We have attempted to develop an approach utilizing natural processes involved in collateral development, even though these are largely unknown. Thus, bone marrow (BM) is a natural source of multiple factors involved in angiogenesis, including fibroblast growth factors 1 and 2 and vascular endothelial growth factor (VEGF) (11,12). In addition, several mediators known to be involved in hematopoiesis may also regulate angiogenesis (13). These multiple cytokines are secreted by several cellular BM components, including stromal cells and various hematopoietic cells (11,13–15). Finally, BM-derived endothelial progenitor cells may be involved in postnatal extramedullary angiogenesis (16,17).

Therefore, in the current investigation we tested the validity of the following hypothesis: "In a porcine coronary occlusion model, direct delivery of freshly aspirated autologous BM (ABM) cells into ischemic myocardium enhances collateral flow delivery and improves myocardial function." We also sought to define whether the BM cells obtained under the specific conditions employed in this investigation secreted potent angiogenic cytokines that may be important cofactors involved in collateral function in ischemic myocardium.

	Methods

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In vitro studies.   Pig BM culture
Bone marrow cells were harvested from pigs with chronic myocardial ischemia and filtered sequentially using 300µ and 200µ stainless steel mesh filters. Bone marrow cells were then isolated by Ficoll-Hypaque gradient centrifugation and cultured in long-term culture medium ([LTCM] Stem Cell Tech, Vancouver, British Columbia, Canada) at 33°C with 5% CO₂. Weekly, half of the medium was removed and replaced with fresh LTCM. The removed medium was filtered (0.2µ filter) and stored at –20°C for subsequent enzyme-linked immunosorbent assay (ELISA) and cell proliferation assays.

Isolation and culture of pig aortic endothelial cells (PAEC)
Fresh PAEC were isolated as previously described (18) and cultured using standard techniques. Their identity was confirmed by typical endothelial cell morphology and by positive antifactor VIII staining.

Effects of conditioned medium on PAEC proliferation and DNA synthesis
Pig aortic endothelial cells (passages 3 to 10) were transferred to 96-well culture plates at a density of 5,000 cells/well. Cells were cultured for two to three days before being used in proliferation and DNA synthesis experiments. The conditioned medium of BM cells cultures, collected at four weeks, were pooled from seven culture flasks and used in the assay. Aliquotes (10 µL, 30 µL, 100 µL or 200 µL) of pooled conditioned medium or LTCM (200 µL, as control) were added to confluent PAEC in 96 well plates in triplicate. Four days after culture with conditioned medium or control medium, the PAEC were trypsinized and counted (Coulter Counter Beckman Corporation, Miami, Florida). DNA synthesis was assayed at two days after the same culture conditions. One µCi tritiated thymidine was added to each well. Forty-eight hours later, DNA in PAEC was harvested (Mach III M Tomtec, Hamden, Connecticut); radioactivity was counted by liquid scintillation counter (Multidetector Liquid Scintillation Luminescence Counter EG&G Wallac, Turku, Finland).

Determination of VEGF and macrophage chemoattractant protein-1 (MCP-1) in conditioned medium
The concentration of VEGF in conditioned medium was measured using sandwich ELISA kit (Chemicon International Inc., Temecula, California), and MCP-1 was assayed by sandwich enzyme immunoassay kit (R & D Systems, Minneapolis, Minnesota) according to the manufacturer’s directions.

In vivo study
The animal study was approved by the Animal Care and Use Committee of the MedStar Research Institute. The protocol conformed to the tenets of the American Heart Association on research animal use. Fourteen specific-pathogen-free domestic pigs weighing approximately 35 to 40 kg underwent ameroid constrictor implantation around the proximal left circumflex artery. Four weeks later, all pigs underwent: 1) selective left and right coronary angiography, 2) transthoracic echocardiography, 3) regional myocardial blood flow assessment, and 4) electromechanical left ventricular mapping. Following these procedures, all animals received morphine sulfate 3 mg intramuscularly every 8 h to alleviate postprocedural related discomfort.

Treatment groups
In a pilot study, we evaluated the feasibility and safety of transendocardial injection of ABM using a tip-deflecting injection catheter (Biosense-Webster, Diamond Bar, California) in 10 ischemic pigs. Each injection site was marked by adding Fluoresbrite YG 2.0 µm microspheres (Polysciences, Inc. Warrington, Pennsylvania) to ABM in a 1 to 9 ratio. Ten injections of 0.2 ml were evenly distributed approximately 1 cm apart, within the ischemic region and its boundaries (lateral wall, n = 5) and within the nonischemic territory (anterior-septal wall, n = 5). Animals were sacrificed at 1, 3, 7 and 21 days (n = 2 in each time point). Two additional animals were also sacrificed at three weeks after 0.5 ml of ABM injections.

In the second phase, animals were randomized to receive 12 injections of 0.2 ml each of freshly harvested ABM aspirate (n = 7) or a similar volume of heparinized saline (n = 7) directed to the ischemic area and its boundaries in a similar fashion to the pilot study. Heart rate and systemic blood pressure were measured continuously, and left atrial pressure was recorded during the myocardial blood flow studies.

An additional seven animals without myocardial ischemia were studied to determine whether transendocardial injection of ABM into normal myocardium increases regional blood flow. Animals were randomized to injections of ABM (n = 4) or heparinized saline (n = 3) into the lateral wall as described above.

BM aspiration and preparation
Immediately after completion of the baseline assessment, animals underwent BM aspiration from the left femoral shaft using standard techniques. Bone marrow was aspirated from two sites (3 ml per site) using preservative-free heparinized glass syringes (20 U heparin/1 ml fresh BM). The bone marrow was immediately macrofiltered using 300µ and 200µ stainless steel filters, sequentially.

Left ventricular mapping procedure
The procedure has been described in detail (19,20). The reconstructed left ventricular electromechanical maps guided direct transmyocardial injections using previously described electromechanical coupling parameters (21).

Echocardiography study
Transthoracic echocardiography (Hewlett-Packard Sonos 1000) image views at the midpapillary muscle level were recorded at rest and during 2 min of right atrial pacing (temporary electrode, 180/min); fractional shortening measurements were obtained as described (21). After being digitized and stored in a frame-grabber board (Kodak ImageVue Compact Version 3.02, Eastman Kodak Company, Pennsylvania), the average of three measurements (taken from three different late-recorded frames) in each examined region was used for analysis.

Angiographic collateral flow assessment
Each coronary angiography study included dedicated injections to detect late collateral dependent filling of the occluded left circumflex artery or its major branches. Angiographic collateral flow was assessed using the Rentrop semiquantitative (0 to 3) score system for both left to left and right to left selective coronary injection (22).

Regional myocardial blood flow
Fluorescent colored microspheres (23) (Interactive Medical Technologies, West Los Angeles, California) were used to assess regional myocardial blood flow at rest and during maximal coronary vasodilation induced by infusing adenosine at a constant rate of 140 µg/kg/min (Fujisawa USA, Deerfield, Illinois). Measurements were quantified by the reference sample technique (24). Two central 7-mm thick slices were each divided into 16 endocardial (n = 8) and epicardial (n = 8) subsegments, as previously described (3,4). The average of eight lateral ischemic zone and eight septal normal zone subsegments measurements was used to assess endocardial and epicardial regional myocardial blood flow. Relative collateral flow was also computed as described (3).

Histopathology
Standard BM smears were prepared before and after propelling freshly filtered ABM aspirate through the needle using similar injecting pressure as in the in vivo study.

In the pilot study, identified fluorescent-labeled areas were cut into three full thickness adjacent blocks, immersion-fixed and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff. Fluorescent-labeled tissues obtained from the ischemic region were snap-frozen and stained for CD-34, a marker of BM-derived progenitor cells, using monoclonal mouse antibody as primary antibody (Becton Dickinson, San Jose, California).

In the efficacy study phase, full-thickness, 1.5 cm³ sections from ischemic and nonischemic regions were processed for paraffin sections. Each sample was stained with H&E, Masson’s trichrome and factor VIII related antigen. Vascularity, including total area occupied by any blood vessel, the number of these vessels and those with a diameter larger than 50 µm, was assessed, as previously described (25).

Density of endothelial cells was assessed using Sigma-Scan Pro morphometry software (SPSS Science, Chicago, Illinois) and the intensity threshold method. Total endothelial area for each sample and for each specimen was obtained along with relative percent endothelial area (endothelial area/area of myocardium studied). Similar analysis was performed for noninfarcted (viable) myocardium after excluding trichrom-positive stained areas.

Statistics
Unpaired and paired Student t tests were used for comparison of nominal and normalized values between and within groups, respectively. Wilcoxon rank-sum test was used when test for normal distribution failed. Repeated measure of variance was used to compare the concentration of VEGF and MCP-1 in the control medium or BM-conditioned medium collected at weeks 1, 2, 3 and 4. One-way analysis of variance (ANOVA) was used to compare the effects of conditioned medium on PAEC proliferation and tritiated thymidine uptake by PAEC. All results are presented as mean ± SD. A p value <0.05 was considered as statistically significant.

	Results

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In vitro study.   Conditioned medium-induced proliferation of porcine aortic endothelial cells
The BM-conditioned medium collected at four weeks increased proliferation of PAEC as determined by both direct counting and measuring tritiated thymidine uptake (p < 0.001 for both; Fig. 1, A and B) in a dose-related manner. When LTCM was completely replaced by BM-conditioned medium (200 µL), the observed stimulatory effect diminished. Similar dose-related results were observed in the tritiated thymidine uptake studies.

View larger version (27K): [in this window] [in a new window]   Figure 1 (A) Effects of conditioned medium on PAEC proliferation. Pig aortic endothelial cells were seeded, in triplicate, in 96 well plates for one day; subsequently, the indicated volumes of conditioned medium from pooled samples or control medium were added (with comparable volume of PAEC medium removed). After four days, PAEC, cultured with conditioned medium or control medium, were harvested and counted. (B) Effects of conditioned medium on tritiated thymidine uptake by PAEC. After two days, 1 µCi tritiated thymidine was added to each well, and 48 h later, DNA in PAEC was harvested, and radioactivity was counted. (C and D) Changes in bone marrow cell conditioned medium of VEGF (C) and MCP-1 (D) concentrations at one to four weeks, assayed by ELISA. MCP-1 = macrophage chemoattractant protein-1; PAEC = pig aortic endothelial cells; VEGF = vascular endothelial growth factor.

VEGF and MCP-1 in BM-conditioned medium and in control medium
Over four weeks, concentrations of VEGF and MCP-1 in BM-conditioned medium increased gradually to seven and three times the first week level, respectively (Fig. 1, C and D). Vascular endothelial growth factor and MCP-1 levels in a control culture medium, not exposed to BM, were 0 and 11 ± 2 pg/ml, respectively.

In vivo study
Fourteen of 20 pigs completed the study. At intervention, weight was similar between groups (ABM vs. controls = 68 ± 4 vs. 69 ± 4 kg). Five died suddenly within three weeks of ameroid implantation (before ABM injection), presumably from ameroid-induced abrupt coronary occlusion. All animals survived the BM aspiration and catheter-based intramyocardial injections. One animal, randomized to the ABM-treated group, experienced acute myocardial ischemia during ameroid implantation and died seven days before study termination. All animals were hemodynamically stable, and no differences were noted between groups in heart rate, mean blood pressure or mean left atrial pressures at baseline and at four-week follow-up (data not shown).

Myocardial function
Preintervention relative fractional wall thickening, at rest and during pacing, was similar between groups (p = 0.86 and 0.96, respectively; Table 1). At four weeks, regional wall thickening improved at rest and during pacing, due to an approximately 50% increase in wall thickening of the ischemic lateral wall. No significant changes were observed in the control animals.

Angiographic assessment of collateral flow
Baseline left to left and right to left collateral scoring was similar between groups. At four weeks, no significant improvement was noted in either of the two groups (ABM left to left collaterals: 1.3 ± 1.2 vs. 1.4 ± 1.0 at four weeks, p = 1.0 and right to left collaterals 0.7 ± 0.5 vs. 1.0 ± 1.2, p = 0.46; control: 2.0 ± 1.2 vs. 2.4 ± 0.8, p = 0.25 and right to left collaterals 0.9 ± 1.1 vs. 0.6 ± 0.8, p = 0.46).

Histopathology and vascularity assessment
Bone marrow smears before and after passing the filtrated aspirate through the injection catheter revealed normal morphology, absence of macroaggregates and no evidence of cell fragments or distorted cell shapes. Histopathology at day 1 after injections revealed acute lesions characterized by a needle tract containing fibrin and mononuclear cells that morphologically could not be differentiated from BM cells. Cellularity was maximal at three and seven days and declined subsequently over time. At three weeks, more fibrosis was seen in the 0.5-ml injection sites compared with the 0.2-ml sites. Overall, approximately 4% to 6% of the cellular infiltrate showed positive immunoreactivity to CD-34 (Fig. 2).

View larger version (80K): [in this window] [in a new window]   Figure 2 Myocardial injection site stained for CD-34 (left) and hematoxylin and eosin (right) at three days after transendocardial injection of ABM. Note strong staining for CD-34 in 4% to 6% of the cells.

	Discussion

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We found that direct delivery of freshly aspirated ABM into ischemic porcine myocardium enhances collateral flow and improves myocardial function. The concordant changes in these two separately measured, but functionally dependent, parameters of collateral flow adds further support to the conclusion that injection of ABM into ischemic tissue initiates and sustains biologically relevant angiogenic responses. No change in collateral flow was observed when ABM was injected into normal nonischemic myocardium.

In vitro study.   In these experiments, we found several possible contributing factors that may mediate BM cell-induced increase in collateral flow. First, BM cells in culture produced large amounts of VEGF, a potent angiogenic growth factor (3,7,10), and MCP-1, which has been identified as a mediator of arteriogenesis, a process whereby remodeling of preexisting larger-sized blood vessels occurs, increasing their capacity to deliver blood (26). In addition, the conditioned medium of BM cells (containing secreted products of the cells) induced proliferation of vascular endothelial cells in a dose-related manner. Whether this effect was caused solely by the increased amounts of VEGF and MCP-1 measured or was also contributed to by other, as yet unknown, molecules is uncertain. In addition, we found in vitro a substantial time-related increase in the number and proportion of BM-derived endothelial cells. It is possible that proliferating endothelial cells derived from the injected ABM contributed to collateral development via incorporation into expanding collateral vessels (16); this concept, however, is still controversial (27). Finally, it is possible that the mild transient inflammatory effect we observed to occur the first few days after the injection of the BM cells may have contributed to the initiation of the angiogenic process.

In vivo studies.   The substantial increase in myocardial perfusion we observed was not accompanied by histological evidence of an increased number of capillaries or large blood vessels. Such a discrepancy, also encountered in previous studies (28,29), undoubtedly reflects the very small increases in diameter necessary to effect biologically significant decreases in resistance to collateral flow and the sensitivity of the histologic and angiographic measures we employ to detect such changes, even with detailed methods of angiographic analysis (30). In addition, in the ameroid pig model, standard angiographic assessment may be suboptimal, as extracardiac collaterals, which are not assessed by conventional angiography, account for up to 30% of collateral flow at rest (31).

Catheter-based transendocardial injection had previously been used successfully to deliver solutions such as methylene blue (32) or reporter genes (33), but not cells. The normal BM smears, the positive intramyocardial CD-34 staining at three days after ABM injection and the effects on collateral flow indicate that such an approach does not compromise the function of injected cells. Our study, therefore, establishes, for the first time, the feasibility of a catheter-based transendocardial approach for direct myocardial cell transplantation, a far less invasive procedure than currently available surgical techniques (27).

The evidence that freshly aspirated ABM transplantation into ischemic myocardium is associated with improved collateral flow without adverse effects in the pig model of myocardial ischemia suggests that this strategy may be of clinical importance. Such a strategy would also probably be less costly than many other currently tested angiogenic strategies and would avoid potential toxicity-related issues that, although remote, are definite possibilities with various gene-based approaches using viral vectors.

In summary, the results of this investigation indicate that BM cells secrete potent angiogenic cytokines such as VEGF and MCP-1 and induce proliferation of BM-derived, as well as vascular, endothelial cells. They also show that catheter-based transendocardial injection of ABM is feasible, a strategy resulting in enhanced collateral perfusion and improved mechanical function of ischemic myocardium. These results suggest that clinical studies may now be appropriate to determine whether this novel therapeutic strategy may be an effective way to achieve therapeutic enhancement of collateral development.

	Acknowledgments

 
The authors would like to thank Ms. Jill Walker, BSc, for her excellent technical assistance in conducting the ELISA tests and for Biosense-Webster, Johnson and Johnson, for supplying the injection catheters.

	Footnotes

 
Supported by grants from the Cardiovascular Research Foundation, the Pergament Cardiovascular Research Center, Washington Hospital Center, Washington, DC.

	References

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1. Waltenberger J. Modulation of growth factor action: implication for the treatment of cardiovascular diseases. Circulation. 1997;96:4083–4094[Abstract/Free Full Text]
2. Unger EF, Banai S, Shou M, et al. Basic fibroblast growth factor enhances myocardial collateral flow in a canine model. Am J Physiol. 1994;266:H1588–H1595[Medline]
3. Banai S, Jaklitsch MT, Shou M, et al. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. 1994;89:2183–2189[Abstract/Free Full Text]
4. Lazarous DF, Scheinowitz M, Shou M, et al. Effect of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart. Circulation. 1995;91:145–153[Abstract/Free Full Text]
5. Lazarous DF, Shou M, Scheinowitz M, et al. Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury. Circulation. 1996;94:1074–1082[Abstract/Free Full Text]
6. Giordano FJ, Ping P, Mckirnan D, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nature Med. 1996;2:534–539[CrossRef][Medline]
7. Rosengart TK, Lee LY, Patel SR, et al. Angiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease. Circulation. 1999;100:468–474[Abstract/Free Full Text]
8. Laham RJ, Sellke FW, Edelman ER, et al. Local perivascular delivery of basic fibroblast growth factor in patients undergoing coronary bypass surgery: results of a phase I randomized, double-blind, placebo-controlled trial. Circulation. 1999;100:1865–1871[Abstract/Free Full Text]
9. Schumacher B, Pecher P, von Specht BU, Stegmann T. Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease. Circulation. 1998;97:645–650[Abstract/Free Full Text]
10. Holash J, Wiegand SJ, Yancopoulos GD. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene. 1999;18:5356–5362[CrossRef][Medline]
11. Mohle R, Green D, Moore MA, et al. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA. 1997;94:663–668[Abstract/Free Full Text]
12. Bikfalvi A, Han ZC. Angiogenic factors are hematopoietic factors and vice versa. Leukemia. 1994;8:523–529[Medline]
13. Sensebe L, Deschaseaux M, Li J, et al. The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment. Stem Cells. 1997;15:133–143[Abstract/Free Full Text]
14. Rohde D, Wickenhauser C, Denecke S, et al. Cytokine release by human bone marrow cells: analysis at the single cell level. Virchows Arch. 1994;424:389–395[Medline]
15. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276:71–74[Abstract/Free Full Text]
16. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999;85:221–228[Abstract/Free Full Text]
17. Asahara T, Takahashi T, Masuda H, et al. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J. 1999;18:3964–3972[CrossRef][Medline]
18. Watier H, Vallee I, Thibault G, et al. Effect of human inflammatory cytokines on porcine endothelial cell MHC molecule expression: unique role for TNF-alpha in MHC class-II induction. Transplant Proc. 1994;26:1152–1155[Medline]
19. Kornowski R, Hong MK, Gepstein L, et al. Preliminary animal and clinical experiences using an electromechanical mapping procedure to distinguish infarcted from healthy myocardium. Circulation. 1998;98:1116–1124[Abstract/Free Full Text]
20. Gepstein L, Goldin A, Lessick J, et al. Electromechanical characterization of chronic myocardial infarction in the canine coronary occlusion model. Circulation. 1998;98:2055–2064[Abstract/Free Full Text]
21. Fuchs S, Kornowski R, Shiran A, et al. Electromechanical characterization of myocardial hibernation in a pig model. Coron Artery Dis. 1999;10:195–198[Medline]
22. Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in collateral channel filling immediately after controlled coronary artery occlusion by an angioplasty balloon in human subjects. J Am Coll Cardiol. 1985;5:587–592[Abstract]
23. Glenny RW, Bernard S, Brinkley M. Validation of fluorescent-labeled microspheres for measurement of regional organ perfusion. J Appl Physiol. 1993;74:2585–2597[Abstract/Free Full Text]
24. Heymann MA, Payne BD, Hoffman JI, Rudolph AM. Blood flow measurements with radionuclide-labeled particles. Prog Cardiovasc Dis. 1977;20:55–79[CrossRef][Medline]
25. Hamilton PW. Stereology. Hamilton PW, Allen DC. Quantitative Clinical Pathology. Oxford: Blackwell Science Ltd; 1995. p. 3–15
26. Ito WD, Arras M, Winkler B, et al. Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion. Circ Res. 1997;80:829–837[Abstract/Free Full Text]
27. Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation. 1999;100(Suppl):II247–II256[Medline]
28. Devlin GP, Fort S, Yu E, et al. Effect of a single bolus of intracoronary basic fibroblast growth factor on perfusion in an ischemic porcine model. Can J Cardiol. 1999;15:676–682[Medline]
29. Laughlin MH, Tomanek RJ. Myocardial capillarity and maximal capillary diffusion capacity in exercise-trained dogs. J Appl Physiol. 1987;63:1481–1486[Abstract/Free Full Text]
30. Gibson CM, Ryan K, Sparano A, et al. Angiographic methods to assess human coronary angiogenesis. Am Heart J. 1999;137:169–179[CrossRef][Medline]
31. White FC, Carroll SM, Magnet A, Blooe CM. Coronary collateral development in swine after coronary artery occlusion. Circ Res. 1992;71:1490–1500[Abstract/Free Full Text]
32. Kornowski R, Fuchs S, Tio FO, et al. Evaluation of the acute and chronic safety of the biosense injection catheter system in porcine hearts. Cathet Cardiovasc Intervent. 1999;48:447–453[CrossRef][Medline]
33. Vale PR, Losordo DW, Tkebuchava T, et al. Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping. J Am Coll Cardiol. 1999;34:246–254[Abstract/Free Full Text]

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				R. G. Shaffer, S. Greene, A. Arshi, G. Supple, A. Bantly, J. S Moores, M. S Parmacek, and E. R Mohler III Effect of acute exercise on endothelial progenitor cells in patients with peripheral arterial disease Vascular Medicine, November 1, 2006; 11(4): 219 - 226. [Abstract] [PDF]

				C. Kubal, K. Sheth, B. Nadal-Ginard, and M. Galinanes Bone marrow cells have a potent anti-ischemic effect against myocardial cell death in humans. J. Thorac. Cardiovasc. Surg., November 1, 2006; 132(5): 1112 - 1118. [Abstract] [Full Text] [PDF]

				I. Ben-Dor, S. Fuchs, and R. Kornowski Potential Hazards and Technical Considerations Associated With Myocardial Cell Transplantation Protocols for Ischemic Myocardial Syndrome J. Am. Coll. Cardiol., October 17, 2006; 48(8): 1519 - 1526. [Abstract] [Full Text] [PDF]

				K. Lunde, S. Solheim, S. Aakhus, H. Arnesen, M. Abdelnoor, T. Egeland, K. Endresen, A. Ilebekk, A. Mangschau, J. G. Fjeld, et al. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N. Engl. J. Med., September 21, 2006; 355(12): 1199 - 1209. [Abstract] [Full Text] [PDF]

				M. Saeed, A. J. Martin, R. J. Lee, O. Weber, D. Revel, D. Saloner, and C. B. Higgins MR Guidance of Targeted Injections into Border and Core of Scarred Myocardium in Pigs Radiology, August 1, 2006; 240(2): 419 - 426. [Abstract] [Full Text] [PDF]

				M. Boodhwani, Y. Nakai, P. Voisine, J. Feng, J. Li, S. Mieno, B. Ramlawi, C. Bianchi, R. Laham, and F. W. Sellke High-Dose Atorvastatin Improves Hypercholesterolemic Coronary Endothelial Dysfunction Without Improving the Angiogenic Response Circulation, July 4, 2006; 114(1_suppl): I-402 - I-408. [Abstract] [Full Text] [PDF]

				D. Kaigler, P.H. Krebsbach, Z. Wang, E.R. West, K. Horger, and D.J. Mooney Transplanted endothelial cells enhance orthotopic bone regeneration. J. Dent. Res., July 1, 2006; 85(7): 633 - 637. [Abstract] [Full Text] [PDF]

				H. Hasegawa, H. Takano, K. Iwanaga, M. Ohtsuka, Y. Qin, Y. Niitsuma, K. Ueda, T. Toyoda, H. Tadokoro, and I. Komuro Cardioprotective Effects of Granulocyte Colony-Stimulating Factor in Swine With Chronic Myocardial Ischemia J. Am. Coll. Cardiol., February 21, 2006; 47(4): 842 - 849. [Abstract] [Full Text] [PDF]

				L. Ye, H. K. Haider, and E. K. W. Sim Adult Stem Cells for Cardiac Repair: A Choice Between Skeletal Myoblasts and Bone Marrow Stem Cells Experimental Biology and Medicine, January 1, 2006; 231(1): 8 - 19. [Abstract] [Full Text] [PDF]

				I. Dimarakis, N. A. Habib, and M. Y.A. Gordon Adult bone marrow-derived stem cells and the injured heart: just the beginning? Eur. J. Cardiothorac. Surg., November 1, 2005; 28(5): 665 - 676. [Abstract] [Full Text] [PDF]

				S. Sartore, M. Lenzi, A. Angelini, A. Chiavegato, L. Gasparotto, P. D. Coppi, R. Bianco, and G. Gerosa Amniotic mesenchymal cells autotransplanted in a porcine model of cardiac ischemia do not differentiate to cardiogenic phenotypes Eur. J. Cardiothorac. Surg., November 1, 2005; 28(5): 677 - 684. [Abstract] [Full Text] [PDF]

				B. E. Strauer, M. Brehm, T. Zeus, T. Bartsch, C. Schannwell, C. Antke, R. V. Sorg, G. Kogler, P. Wernet, H.-W. Muller, et al. Regeneration of Human Infarcted Heart Muscle by Intracoronary Autologous Bone Marrow Cell Transplantation in Chronic Coronary Artery Disease: The IACT Study J. Am. Coll. Cardiol., November 1, 2005; 46(9): 1651 - 1658. [Abstract] [Full Text] [PDF]

				R. R. Makkar, M. J. Price, M. Lill, M. Frantzen, K. Takizawa, T. Kleisli, J. Zheng, S. Kar, R. McClelan, T. Miyamota, et al. Intramyocardial Injection of Allogenic Bone Marrow-Derived Mesenchymal Stem Cells Without Immunosuppression Preserves Cardiac Function in a Porcine Model of Myocardial Infarction Journal of Cardiovascular Pharmacology and Therapeutics, October 1, 2005; 10(4): 225 - 233. [Abstract] [PDF]

				V. J. Dzau, M. Gnecchi, A. S. Pachori, F. Morello, and L. G. Melo Therapeutic Potential of Endothelial Progenitor Cells in Cardiovascular Diseases Hypertension, July 1, 2005; 46(1): 7 - 18. [Abstract] [Full Text] [PDF]

				H. K. Haider and M. Ashraf Bone marrow stem cell transplantation for cardiac repair Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2557 - H2567. [Abstract] [Full Text] [PDF]

				V. Schachinger and A. M. Zeiher Stem Cells and Cardiovascular and Renal Disease: Today and Tomorrow J. Am. Soc. Nephrol., March 1, 2005; 16(3_suppl_1): S2 - S6. [Abstract] [Full Text] [PDF]

				K. C. Wollert and H. Drexler Clinical Applications of Stem Cells for the Heart Circ. Res., February 4, 2005; 96(2): 151 - 163. [Abstract] [Full Text] [PDF]

				G. V. Silva, S. Litovsky, J. A.R. Assad, A. L.S. Sousa, B. J. Martin, D. Vela, S. C. Coulter, J. Lin, J. Ober, W. K. Vaughn, et al. Mesenchymal Stem Cells Differentiate into an Endothelial Phenotype, Enhance Vascular Density, and Improve Heart Function in a Canine Chronic Ischemia Model Circulation, January 18, 2005; 111(2): 150 - 156. [Abstract] [Full Text] [PDF]