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Ann Thorac Surg 2009;88:418-424. doi:10.1016/j.athoracsur.2009.04.105
© 2009 The Society of Thoracic Surgeons
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Original Articles: General Thoracic

Syngeneic Bone Marrow Mononuclear Cells Improve Pulmonary Arterial Hypertension Through Vascular Endothelial Growth Factor Upregulation

Homare Yoshida, MD, Takashi Kitaichi, MD, PhD, Masahisa Urata, MD, Hirotsugu Kurobe, MD, PhD, Tamotsu Kanbara, MD, Tatsuo Motoki, MD, Tetsuya Kitagawa, MD, PhD*

Department of Cardiovascular Surgery, University of Tokushima Graduate School, Tokushima, Japan

Accepted for publication April 27, 2009.

* Address correspondence to Dr Kitagawa, Department of Cardiovascular Surgery, University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima, 770-8503, Japan (Email: kitagawa{at}clin.med.tokushima-u.ac.jp).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: We investigated the effects and possible mechanism of syngeneic bone marrow mononuclear cell (BM-MNC) transplantation on pulmonary arterial hypertension induced by monocrotaline.

Methods: Monocrotaline (80 mg/kg body weight) was administrated to C57BL/6 mice, and pulmonary arterial hypertension was induced 4 weeks later. Bone marrow mononuclear cells harvested from syngeneic donor mice were injected intravenously into those mice 4 weeks after monocrotaline administration. The ratio of right ventricular to septum plus left ventricular weight, the number of small pulmonary arteries, and medial thickness of pulmonary arteries were measured. Western immunoblotting of the lung tissue was performed to observe vascular endothelial growth factor and its receptor expression 1 week after BM-MNC transplantation. Vascular endothelial growth factor receptor-2 inhibitor was administered to pulmonary arterial hypertension mice simultaneously with BM-MNC transplantation.

Results: The ratio of right ventricular to septum plus left ventricular weight increased, the number of pulmonary arteries decreased, and medial thickness increased significantly 4 weeks after monocrotaline injection compared with those of vehicle-injected mice. These indices of monocrotaline-injected mice improved significantly 4 weeks after BM-MNC transplantation compared with those of mice at 8 weeks after monocrotaline injection (0.22 ± 0.02 versus 0.31 ± 0.02; 17.1 ± 2.6 versus 8.2 ± 1.7; 7.7% ± 2.2% versus 20% ± 2.1%, respectively; p < 0.01). However, BM-MNCs were not incorporated into the lung at 1 week after transplantation, and significant vascular endothelial growth factor upregulation and without receptor expression was observed in lung tissue 1 week after transplantation. Improvement of pulmonary arterial hypertension was inhibited by simultaneous administration of vascular endothelial growth factor receptor-2 inhibitor with BM-MNC transplantation.

Conclusions: These results indicate that syngeneic BM-MNC transplantation improves monocrotaline-induced pulmonary arterial hypertension by favorable pulmonary artery remodeling through vascular endothelial growth factor upregulation.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Pulmonary arterial hypertension (PAH) is characterized by high pulmonary arterial pressure, increased pulmonary vascular resistance, right ventricular (RV) hypertrophy, and RV failure. Primary PAH is a fatal disorder of unknown pathogenesis, and secondary PAH associated with several heart and lung diseases sometimes leads to devastating outcomes. Although several advancements in the pharmacologic field have been devoted to the treatment of PAH in the recent decade, PAH still has only a few satisfactory long-term treatments.

It is known that circulating bone marrow (BM)-derived progenitor cells play an important role in the repair of endothelial injury and postnatal angiogenesis [1–3]. In 2001, Campbell and colleagues [4] reported cell-based vascular endothelial growth factor (VEGF) gene transfer as a neovascularization therapy for PAH. Since then, diverse basic research has been performed to prevent pathologic arterial remodeling in PAH animal model, particularly by intravenous transplantation of BM-derived cells with or without gene therapy [5, 6]. However, exogenous and genetically modified BM-derived cells are rarely incorporated into the injured endothelium of remodeled pulmonary arterioles [1, 3]. Other studies have suggested that only the upregulation of some tissue cytokines like classic angiogenic growth factors consequent to the transplantation of BM-derived cells could restore damaged vessels [7–10]. The increase in survival of the endothelial progenitor cells and potential paracrine mechanisms provided by the local secretion of VEGF may underlie neovascularization. Thus, different or contrary perspectives related to the efficacy of diversified assessments and their mechanisms are still a matter of debate.

Our present aim is to elucidate the efficacy of intravenous transplantation of syngeneic BM mononuclear cells (BM-MNCs) as a therapeutic option for PAH and the mechanism of favorable pulmonary artery remodeling through VEGF and its receptor system.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Animal Preparation
Wild-type C57BL/6 female mice (Japan SLC, Tokyo, Japan) were used at 8 weeks of age and were anesthetized with ketamine and xylazine. Animal care and procedures were in accordance with the institutional guidelines.

Pulmonary Arterial Hypertension Models
Pulmonary endothelial toxin monocrotaline (C16H26O6N; Matrix Scientific, Columbia, SC) treatment was previously demonstrated as capable of inducing PAH in rats [3, 6]. A single injection of monocrotaline (5, 30, 60, 80, or 120 mg/kg) into the peritoneum was performed. An appropriate dose of monocrotaline for a PAH mouse model like the PAH rat model was determined to be 80 mg/kg for the completion rate of PAH lesion of 90% to 100% with a survival rate of 80%, 4 weeks after the intraperitoneal monocrotaline administration. That dosage did not cause toxic effects to the liver. We made a murine PAH model within 4 weeks by intraperitoneal 80 mg/kg monocrotaline administration in spite of a slightly lower grade of PAH than the PAH rat model, the same as the hypoxia-induced PAH mouse model (M Urata, H Yoshida, and T Kitagawa, unpublished data).

Isolation of Bone Marrow Mononuclear Cells and Intravenous Transplantation
We used unfractionated BM-MNCs in this study. Bone marrow was aspirated from the femurs and tibias of 8-week-old female C57BL/6 mice. Mononuclear cells were isolated by density gradient (Kokusan, Tokyo, Japan) centrifugation at 1,200 rpm for 15 minutes. For cell delivery, BM-MNCs were resuspended in phosphate-buffered saline (PBS) with 1 x 107/0.25 mL of concentration. This suspension of BM-MNCs (1 x 107 cells) was injected into monocrotaline-treated PAH mice through the orbital vein. The number of injected BM-MNCs was based on the previous papers using unfractionated BM cells in a mouse or rat PAH model (5 x 106 to 1 x 108 cells) [2, 11].

Study Protocol
The mice were divided into four groups: (1) vehicle-injected mice (control; n = 10); (2) monocrotaline-treated mice (PAH model-4w; n = 10); (3) monocrotaline-treated mice 4 weeks after BM-MNC transplantation (BMT; n = 10); and (4) monocrotaline-treated mice 4 weeks after PBS injection (PAH model-8w; n = 10).

Assessment of Pulmonary Arterial Hypertension
The ratio of right ventricular to ventricular septum plus left ventricular weight (RV/S+LV) was measured by the previously described estimation method of RV hypertension caused by PAH [12].

Histologic Evaluation
The lungs were perfused from the heart with PBS solution and perfusion-fixed with 4% paraformaldehyde in PBS solution. Five-micrometer-thick lung sections were cut and stained with elastica van Gieson and von Willebrand staining after fixation and paraffin embedding. In each mouse, 20 intraacinar vessels accompanying the alveolar ducts or alveoli were examined. The number of small pulmonary arteries (50 to 100 µm in diameter) was counted under a light microscope (x40 magnification) for 20 fields randomly selected. Medial thickness of small pulmonary artery (50 to 100 µm in diameter) was measured and percent medial thickness was calculated as medial thickness divided by diameter of pulmonary artery x100.

Assessment of Migration of Transplanted Bone Marrow Mononuclear Cells Into the Lung Tissue
To investigate whether injected BM-MNCs were incorporated into the lung, labeled BM-MNCs were injected into the monocrotaline-treated mice. All their BM-MNCs were labeled with fluorescent carbocyanine (Di-I; Molecular Probes, Eugene, OR) before cell transplantation. Frozen lung section was made at 3 hours, 24 hours, and 1 week after BM-MNC transplantation, and the degree of cell migration into the lung was assessed under an epifluorescence microscope (Olympus, Tokyo, Japan) [13].

Vascular Endothelial Growth Factor and Its Receptor Expression in the Lung Tissue
To assess the working mechanisms of BM-MNCs, VEGF and its receptor (VEGF receptor-2; Flk-1/KDR) expression in the lung tissue were qualitatively analyzed by Western immunoblotting using specimens obtained from mice 1 week after BM-MNC transplantation and from those of the control and monocrotaline-treated mice. For VEGF and VEGF receptor-2 evaluation, the isolated lungs were quickly frozen in liquid nitrogen. Whole-cell lysates were isolated from the homogenized lung samples and centrifuged at 12,000g for 10 minutes at 4°C to separate the soluble from insoluble fractions. Protein concentration was measured spectrophotometrically at 590-nm wavelength with a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of total protein was fractionated by 10% gradient, sodium dodecylsulfate polyacrylamide gel electrophoresis (Wako Pure Chemical Industries, Osaka, Japan) and transferred to polyvinyl difluoride membranes (GE Healthcare, Pollards Wood, UK). Each membrane was incubated with specific antibodies as follows: anti-VEGF antibody, anti-Flk-1/KDR polyclonal antibody (dilution 1:500; Santa Cruz Biotechnology, Inc, Santa Cruz, CA). Then the membranes were incubated for 1 hour in diluted appropriate secondary antibody (GE Healthcare). Immune complexes were visualized with the enhanced chemiluminescence detection system (GE Healthcare). Bands were quantified by densitometry of the radioautograph films.

Administration of Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor
To completely confirm the beneficial effects of upregulation of VEGF, we administrated VEGF receptor-2 tyrosine kinase inhibitor (C18H18N2O3: (Z)-3-[(2,4-dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indolin-2-one; concentration at which 50% inhibition occurs, 70 nmol/L; Calbiochem, San Diego, CA) [14] to the PAH model mice simultaneously with BMT (BMT + VEGF receptor-2 inhibitor group, n = 10). The description of the chemical substance is highly selective for murine VEGF receptor-2, soluble with dimethyl sulfoxide (10 mg/mL; Fisher Scientific Co, Pittsburgh, PA), and cell permeable. The administration dose of the inhibitor was decided from the concentration at which 50% inhibition occurs and the predictive value of water content in the murine body. Vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor (300 µg) was initially administered subcutaneously on the same day of BM-MNC transplantation, and a dose of 100 µg was given every 2 days for 2 weeks (day 1, 2, 4, 6, 8, 10, 12, 14).

Statistical Analysis
Data are presented as the mean ± standard deviation. Statistical comparisons were performed using unpaired two-tailed Student's t tests or analysis of variance with Scheffe's test as appropriate, with a probability value of less than 0.05 taken to indicate significance.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
The medial wall of the distal small pulmonary artery was thickened significantly, and remarkable fibrosis of the adventitia was observed in the PAH mice (Fig 1B) compared with that of the control mice (Fig 1A). However, these findings improved 4 weeks after BM-MNC transplantation (Fig 1C). The number of small pulmonary arteries decreased significantly in the PAH model (Fig 1F), compared with that of the control mice (Fig 1E), and recovered up to the same level as that in the control mice after BM-MNC transplantation (Fig 1G).


Figure 1
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  		Fig 1. Histoimmunologic findings related to pulmonary artery remodeling. (A) and (E) Control pulmonary vessels. (B) The medial wall of a distal small pulmonary artery is thickened significantly and remarkable fibrosis of adventitia is observed; (F) the number of small pulmonary arteries decreases significantly 4 weeks after monocrotaline injection. Arrows indicate medial thickness. Black bar signifies 50 µm. (C) and (G) Aggravated findings from (B) and (F) improve and recover up to the same level and condition as that in the control mice 4 weeks after syngeneic bone marrow mononuclear cell transplantation. (D) and (H) Among pulmonary arterial hypertension model mice 8 weeks after monocrotaline injection, there was no tendency of spontaneous recovery of pulmonary arterial hypertension. Lung sections are stained either by elastica van Gieson method (A–D; x400) or von Willebrand immunostaining (E–H; x100).

 
Pulmonary arterial hypertension developed within 4 weeks after monocrotaline injection, as shown by a sustained increase in RV/S+LV, as well as muscularization in the media and decrease in the number of distal pulmonary vessels, compared with the vehicle-injected animals. The RV/S+LV ratio was significantly elevated 4 weeks after monocrotaline injection, which is consistent with PAH, compared with that in the vehicle-injected mice (0.29 ± 0.04 versus 0.20 ± 0.02; p < 0.01; Fig 2). The number of pulmonary arteries decreased significantly in the monocrotaline-treated mice compared with that of the vehicle-injected mice (5.1 ± 1.7 versus 20.9 ± 4.8; p < 0.01; Fig 3). Medial thickness also increased significantly in the monocrotaline-treated mice compared with that of the vehicle-injected mice (22.0% ± 5.2% versus 7.5% ± 2.1%; p < 0.01; Fig 4).


Figure 2
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  		Fig 2. Change in the right ventricular to ventricular septum plus left ventricular weight ratio compared with control in pulmonary arterial hypertension (PAH) model 4 weeks (4w) after monocrotaline-injection; bone marrow mononuclear cell transplantation (BMT) model 4 weeks after syngeneic transplantation; pulmonary arterial hypertension model 8 weeks (8w) after monocrotaline injection; and bone marrow mononuclear cell transplantation plus vascular endothelial growth factor (VEGF) receptor-2 inhibitor 4 weeks after vascular endothelial growth factor receptor-2 inhibitor-injection (*p < 0.01; **p < 0.05).

 

Figure 3
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  		Fig 3. Change in the number of small pulmonary arteries compared with control in pulmonary arterial hypertension (PAH) model 4 weeks (4w) after monocrotaline-injection; bone marrow mononuclear cell transplantation (BMT) model 4 weeks after syngeneic transplantation; pulmonary arterial hypertension model 8 weeks (8w) after monocrotaline injection; and bone marrow mononuclear cell transplantation plus vascular endothelial growth factor (VEGF) receptor-2 inhibitor 4 weeks after vascular endothelial growth factor receptor-2 inhibitor-injection (*p < 0.01).

 

Figure 4
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  		Fig 4. Change in the medial thickness of small pulmonary artery compared with control in pulmonary arterial hypertension (PAH) model 4 weeks (4w) after monocrotaline-injection; bone marrow mononuclear cell transplantation (BMT) model 4 weeks after syngeneic transplantation; pulmonary arterial hypertension model 8 weeks (8w) after monocrotaline injection; and bone marrow mononuclear cell transplantation plus vascular endothelial growth factor (VEGF) receptor-2 inhibitor 4 weeks after vascular endothelial growth factor receptor-2 inhibitor-injection (*p < 0.01).

 
Improvement of Pulmonary Arterial Hypertension After Syngeneic Bone Marrow Mononuclear Cell Transplantation
No mice were dead in the acute phase after BM-MNC transplantation and no symptom of thrombotic microangiopathy, which is known as a possible complication of BM-MNC transplantation, was observed. The RV/S+LV ratio was reduced significantly 4 weeks after BM-MNC transplantation compared with that of the monocrotaline plus PBS-treated mice (0.22 ± 0.02 versus 0.31 ± 0.02; p < 0.05; Fig 2). The number of pulmonary arteries increased significantly 4 weeks after BM-MNC transplantation compared with that of the monocrotaline plus PBS-treated mice (17.1 ± 2.6 versus 8.2 ± 1.7; p < 0.01; Fig 3). Medial thickness improved significantly 4 weeks after BM-MNC transplantation compared with that of the monocrotaline plus PBS-treated mice (7.7% ± 2.2% versus 20% ± 2.1%; p < 0.01; Fig 4). Among the group PAH model-8w, the RV/S+LV ratio, the number of pulmonary arteries, and percent medial thickness were all aggravated 8 weeks after monocrotaline injection, and there was no tendency of spontaneous recovery of PAH (Figs 1D, 1H). There was no significant difference between the diameters of the observed pulmonary arteries of each group (control, 79.9 ± 17.8 µm; PAH model, 72.1 ± 23.4 µm; 4 weeks after BMT, 70.0 ± 9.83 µm; 8 weeks after BMT, 68.2 ± 10.6 µm).

Detection of Donor-Derived Bone Marrow Mononuclear Cells in the Recipient Lung
We were unable to detect transplanted cells labeled with the fluorescent carbocyanine in the lung tissue after 3 hours, 24 hours, and 1 week after BM-MNC transplantation.

Vascular Endothelial Growth Factor Concentrations and Vascular Endothelial Growth Factor Receptor-2 Expression in the Lung Tissue
Immunostaining revealed that VEGF expression was significantly enhanced in the lung tissue 1 week after BM-MNC transplantation compared with that of the control and PAH mice (Fig 5). The amount of VEGF expressed in the lung tissue after BM-MNC transplantation was 2.5 times the level before BM-MNC transplantation.


Figure 5
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  		Fig 5. Change in the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 in lung tissue. Western immunoblotting (top) of the lung tissue shows that the expression of vascular endothelial growth factor is enhanced in the lung tissue 1 week after bone marrow mononuclear cell transplantation (BMT-1w). There is no significant difference between the value of vascular endothelial growth factor of lung tissue in control mice and that of the lung tissue 4 weeks after monocrotaline injection (pulmonary arterial hypertension; PAH model-4w). Vascular endothelial growth factor receptor-2 expression (bottom) in the lung tissue 1 week after bone marrow mononuclear cell transplantation is not significantly enhanced and not downregulated compared with those in the control and pulmonary arterial hypertension mice (*p < 0.01).

 
Vascular endothelial growth factor receptor-2 expression in the lung tissue 1 week after BM-MNC transplantation was not significantly enhanced and not downregulated compared with that in the control and PAH mice by Western immunoblotting (Fig 5).

Effects of Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor
In the BMT+VEGF receptor-2 inhibitor group, PAH became worse and reached the same level as that of the PAH model-8w group. The RV/S+LV ratio was still elevated, similar to that of the PAH mice (0.28 ± 0.02; p < 0.01; Fig 2). The number of small pulmonary arteries also decreased (8.1 ± 1.7; p < 0.01; Fig 3), and medial thickness (70.9 ± 18.1 µm in diameter) remained at the same level as that in the PAH mice (21% ± 2.1%; p < 0.01; Fig 4). Beneficial effects of intravenous transplantation of syngeneic BM-MNCs in monocrotaline-injured lung were inhibited and impaired by simultaneous administration of the VEGF receptor-2 inhibitor.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Most patients with severe PAH have shown little favorable response to any medication. A few studies using rat or mouse models in which exogenous and genetically modified BM-derived cells or whole unfractionated BM-derived cells were incorporated into the diseased lung and thereby contributed to repair the pulmonary microvascular structure have been reported in 2005 [2, 3]. However, because it was also suggested in 2007 both that BM-derived cells were rarely incorporated into the diseased lung, in particular the wall of the vessel, and took little participation in the pathologic remodeling of pulmonary arterioles [11], more advanced mechanisms of cell therapies for diseased lung are expected to be elucidated. The BM-MNCs contain multipotent adult endothelial progenitor cells that show a high capacity for differentiation [15].

We projected this study with an expected concept that syngeneic BM-MNC transplantation might bring favorable pulmonary artery remodeling to the diseased lung by activating paracrine angiogenic factors through incorporated BM-MNCs. If so, this favorable pulmonary artery remodeling would be expected to be applicable extensively to out of candidates for definitive repair of the congenital heart defects because of the primitive or injured pulmonary vasculature.

Improvement of Pulmonary Arterial Hypertension After Syngeneic Bone Marrow Mononuclear Cell Transplantation and Whereabouts of Those Cells
We observed remarkable improvements of the RV/S+LV weight ratio as well as the number and medial thickness of small pulmonary arteries after intravenous syngeneic BM-MNC transplantation in the established PAH lesion. These results show that syngeneic BM-MNC transplantation causes favorable pulmonary artery remodeling and improves pulmonary hemodynamics and PAH in mice exposed to monocrotaline. However, there were some controversial data relevant to the efficacy of intravenous transplantation of BM-derived cells in PAH lesion [2–4, 6, 11, 16–18].

With regard to the sources of the transplanted cells, the prior pioneer study demonstrated that circulating endothelial progenitor cells could migrate to injured or ischemic sites and differentiate into mature endothelial cells, finally giving rise to neovascularization in mice [1]. Purified BM-derived progenitor cells greater than 2.5 x 106 limit pulmonary artery remodeling induced by monocrotaline but not by hypoxia in mice, and irradiating those cells had no effect on the development of PAH in monocrotaline-injured mice [6]. Using the rat model, Zhao and colleagues [3] revealed that 1 x 106 BM-derived endothelial progenitor cells could engraft and repair the monocrotaline-damaged lung, restoring microvascular structure and function of monocrotaline-injured rat models. On the other hand, even unfractionated whole BM cells contribute to pulmonary vascular remodeling in the hypoxia-induced PAH mice model [2].

However, recent articles have accumulated different perspectives relevant to transplantation of unfractionated BM-derived cells, namely that the engraftment of BM-derived cells in the lung has rarely been confirmed [11, 16, 17]. O'Neill and associates [18] suggested a potentially beneficial action of unfractionated BM-derived cells during hypoxia through paracrine release of growth factors but not transdifferentiation into endothelial cells. Moreover, we were also unable to detect the transplanted 1 x 107 BM-MNCs in the lung by 1 week after syngeneic BM-MNC transplantation in spite of simultaneous favorable pulmonary artery remodeling.

Not only the different sources and the number of the transplanted cells (ie, circulating BM-derived cells, unfractionated BM cells, or ex vivo expanded endothelial progenitor cells) but also the different species, the experimental methods, and the severity of animal PAH model may account for those discrepancies [19]. As the favorable pulmonary artery remodeling effects were observed in the BM-MNC-transplanted group only, this fact should make us think that these cells play some role in this improvement. Although we failed to detect any of the implanted cells within the lungs or the pulmonary vessels, we cannot rule out the possibility of a technical problem with our detection system. Certain subpopulations of BM-MNCs might be recruited to the lung and could participate in pulmonary artery remodeling. It is necessary to further investigate this possibility to really attribute the effects to BM-MNCs.

Vascular Endothelial Growth Factor and Its Receptor System and Pulmonary Artery Remodeling After Syngeneic Bone Marrow Mononuclear Cell Transplantation
Syngeneic smooth muscle cell–based VEGF gene transfer was effective in preventing the development and progression of PAH in the monocrotaline-induced rat model, which suggested that VEGF was a key regulator of physiologic and pathologic angiogenesis [4]. Our study revealed that VEGF in the lung tissue 1 week after BM-MNC transplantation was upregulated significantly compared with that before BM-MNC transplantation.

The biologic effects of VEGF are mediated by receptor tyrosine kinases. There is now an agreement that VEGF receptor-2 is the major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF [8]. However, our study demonstrated that VEGF receptor-2 expression in the lung tissue 1 week after BM-MNC transplantation was not significantly upregulated or downregulated compared with that of the control and PAH mice. Moreover, VEGF receptor-2 inhibitor itself improves PAH; however, we think that the change will be limited to a low level owing to the slightly upregulated VEGF level associated with the progression of PAH lesion [20].

Oxygen tension plays a key role in regulating the expression of a variety of genes. Hypoxia-inducible factor-1 is a key mediator of hypoxic responses [21]. Vascular endothelial growth factor mRNA expression is induced by exposure to low oxygen tension under a variety of pathophysiologic circumstances [7]. Our recent studies related to this theme revealed that endothelial cells, medial smooth muscle cells, and interstitial cells in monocrotaline-injured lung tissue were suffering the effect of hypoxia (M Urata, H Yoshida, and T Kitagawa, unpublished data). Summing up our results, the activated hypoxia-inducible factor-1 signaling pathway in monocrotaline-injured hypoxic lung tissue could induce significant VEGF-upregulation, which might result in the favorable pulmonary artery remodeling [7–10, 22–24].

Mechanistic Explanation as to How Syngeneic Bone Marrow Mononuclear Cells and Vascular Endothelial Growth Factor Work in Pulmonary Arterial Hypertension Lesion
Monocrotaline-injured pulmonary arterioles and capillaries were frequently occluded by thrombosis in situ with endothelial cell apoptosis [11]. Certain subpopulations of implanted BM-MNCs recruited to the monocrotaline-injured hypoxic pulmonary arterioles, which might stimulate endothelial cells, hypertrophied medial smooth muscle cells, and interstitial cells, could augment local VEGF concentration sufficient for pulmonary artery remodeling in the hypoxic destabilized vessels [25–27]. In particular, an antiapoptosis effect of the augmented VEGF may result in significant improvement of the pulmonary vasculature. A resident population of apoptotic cells that is competent to respond to VEGF may also constitute a potentially limiting factor in these cell therapies designed to promote pulmonary artery remodeling.

Study Limitations
This study has three crucial limitations. First, the working mechanisms of BM-MNC transplantation and VEGF for pulmonary artery remodeling cannot be addressed completely and remains to be elucidated. We have not obtained similar results to those we observed after syngeneic BM-MNC transplantation using administration of VEGF itself to PAH-established mice. Cytokine administration clearly constitutes only one aspect of the therapeutic intervention. What is the most suitable method to treat PAH administration of VEGF—intravenous transplantation of syngeneic BM-MNCs or a combination of both? Second, recent studies reported that angiogenic growth factors were enhanced by transplantation of BM-derived progenitor cells and repair damaged pulmonary arteries. However, how or what BM-derived cells contribute to the vascular arterial remodeling or homeostasis of the lung vasculature after injury is still a matter of debate. For further studies, if we obtain VEGF knockout animals to use as cells donors or recipients, it would allow us to determine whether the source of the VEGF is the transplanted cells themselves, or whether the cells induce other cells in the lung to secrete VEGF. Because only a rodent model was used, if possible, additional experiments using large animals such as pigs or dogs will be closer to the clinical situation for PAH. Third, another important limitation in this study is that we could not determine the cell numbers needed for therapeutic option. Future studies will be needed to determine the dose-dependent effects of implanted BM-MNCs and ascertain its safety and long-term durability.

Conclusions
Syngeneic BM-MNCs might play an important role in the therapeutic pulmonary artery remodeling in lung tissue with pulmonary vascular obstructive disease. The VEGF upregulation by paracrine effect based on certain subpopulations of implanted-BM-MNCs and its receptor system may be one of the potential pathways for the pulmonary artery remodeling in the lung tissue. If the restoration therapy using unpurified populations of BM-MNCs can be used in the human condition with PAH, it will be very useful in treating this disease that has limited long-term therapeutic options.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
We thank Professor Masanori Yoshizumi, MD, Keisuke Ishizawa, PhD, and Narantungalag Dorjsuren, MD, for their excellent contribution related to VEGF receptor-2 expression in the lung tissue.

This study was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (#17591473, #20591649).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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