Hypoxia

Study objective: In these days, it was reported that bone marrow (BM) cells might take part in the remodeling of some systemic vascular diseases; however, it remains unknown whether the BM cells were involved in the vascular remodeling of pulmonary arteries and the progression of pulmonary hypertension (PH). The purpose of this study was to investigate whether BM-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced PH.

Materials and methods: To investigate the role of BM-derived cells, we transplanted the whole BM of enhanced green fluorescent protein (GFP)-transgenic mice to the lethally irradiated syngeneic mice (n = 30). After 8 weeks, chimera mice were exposed to consistent hypoxia using a hypoxic chamber (10% [O.sub.2]) for up to 4 or 8 weeks (10 mice per group). After hemodynamics and the ratio of right ventricular (RV) weight to left ventricle (LV) weight, RV/(LV + septum [S]), were measured, histologic and immunofluorescent staining were performed.

Results: BM-transplanted mice showed a high chimerism (mean [[+ or -] SEM], 91 [+ or -] 2.3%). RV systolic pressure and the RV/(LV + S) ratio increased significantly with time in PH mice, indicating RV hypertrophy. Marked vascular remodeling including medial hypertrophy and adventitial proliferation was observed in the pulmonary arteries of PH mice. Strikingly, a number of GF[P.sup.+] cells were observed at the pulmonary arterial wall, including the adventitia, in hypoxia-induced PH mice, while very few cells were observed in the control mice. Metaspectrometer measurements using confocal laser scanning microscopy confirmed that this green fluorescence was produced by GFP, suggesting that these GF[P.sup.+] cells were mobilized from the BM. Most of them expressed [alpha]-smooth muscle actin, a smooth muscle cell, or myofibroblast phenotype, and contributed to the pulmonary vascular remodeling. A semiquantitative polymerase chain reaction of the GFP gene revealed that the BM-derived GFP-positive cells in the PH group were observed more than eightfold as often compared with the control mice.

Conclusion: The BM-derived cells mobilize to the hypertensive pulmonary arteries and contribute to the pulmonary vascular remodeling in hypoxia-induced PH mice.

Key words: bone marrow transplantation; hypoxia; myofibroblast; progenitor cell; pulmonary hypertension; vascular remodeling

Abbreviations: BM = bone marrow; BMT = bone marrow transplantation; CLSM = confocal laser scanning microscope; FACS = fluorescence-activated cell sorter; GFP = enhanced green fluorescent protein; GF[P.sup.+] [alpha]-SM[A.sup.+] = green fluorescent protein-positive cells that coexpressed [alpha]-smooth muscle actin; LV = left ventricle, ventricular; PCR= polymerase chain reaction; PH = pulmonary hypertension; RV = right ventricle, ventricular; RVSP = right ventricular systolic pressure; S = septum; SMA = smooth muscle actin; SMC = smooth muscle cell

**********

Pulmonary hypertension (PH) is characterized by high pulmonary arterial pressure, increased pulmonary vascular resistance, right ventricular (RV) hypertrophy, and RV failure. The causes of primary PH remain unknown, but it frequently leads to RV failure and death. (1,2) Secondary PH occurs as a complication of pulmonary obstructive disease, lung diseases, and heart failure. Although much effort has been devoted to the treatment of PH, there is still no effective therapy available to prevent it.

The pathogenesis of PH remains unclear, but is likely to at least in part be mediated by hypoxia. Acute exposure to hypoxia induces a selective pulmonary arterial vasoconstriction and an increase in pulmonary arterial pressure. Chronic hypoxia causes sustained PH and pulmonary vascular remodeling. (3) Pulmonary vascular remodeling is characterized by intimal thickening, medial hypertrophy, adventitial proliferation and abnormal extracellular matrix deposition. Medial hypertrophy results from the proliferation of smooth muscle cells (SMCs), and intimal thickening is caused by fibrosis, and the proliferation of endothelial cells, SMCs, and myofibroblasts. (3,4) Proliferating adventitial fibroblasts may be induced by a trigger such as hypoxia to differentiate into myofibroblasts and migrate, which contributes to medial and intimal remodeling. (5-8) The progression of vascular remodeling results in vascular lumen narrowing, increased pulmonary artery resistance, and pulmonary arterial hypertension. However, the precise mechanism of vascular remodeling in PH remains unresolved.

Studies of the pathogenesis of vascular remodeling in general systemic vascular diseases have focused largely on the role of SMCs, which are derived locally from the adjacent medial layer, in vascular remodeling. However, there is accumulating evidence of the existence of circulating progenitor cells such as smooth muscle progenitor cells, endothelial progenitor cells, and fibroblasts. These cells can mobilize to sites of tissue injury and contribute to vascular remodeling in models of vascular disease such as postangioplasty restenosis, graft vasculopathy, and hyperlipidemia-induced atherosclerosis. (9-11)

Recently, it was shown (12) that lung fibroblasts in pulmonary fibrosis could also be derived from bone marrow (BM) progenitor cells using BM chimera mice. In PH, the seminal study of Davie et al (13) raised the concept of BM cell mobilization to hypoxia-induced remodeled pulmonary arteries by demonstrating the existence of c-[kit.sup.+] cells around remodeled pulmonary arteries in hypoxia-induced PH calf lungs. However, a more recent study by Hu et al (14) demonstrated the presence of Sca-[1.sup.+] c-[kit.sup.+] vascular progenitor cells, which were not BM-derived, in vascular adventitia. To clarify whether BM-derived cells can migrate to the hypoxia-induced PH lung, we generated chimeric mice by the transplantation of BM cells from enhanced green fluorescent protein (GFP)-transgenic mice into normal donor mice. We investigated BM cell mobilization to remodeled pulmonary arteries following prolonged exposure to hypoxia using a hypoxic chamber. In this report, we demonstrate directly that BM-derived cells can home to remodeled pulmonary arteries in PH mice and contribute to pulmonary vascular remodeling.

MATERIALS AND METHODS

Mice

C57/BL6 mice were purchased in Japan (Japan CLEA; Tokyo, Japan). GFP mice (C57BL/6 background) were provided by Professor Okabe (Osaka University; Osaka, Japan). (15)

BM Transplantation

GFP mice 8 to 10 weeks old were used as BM donors. Recipient C57BL/6 mice (8 to 10 weeks old) were lethally irradiated with a dose of 8.5 Gy. Five million unfractionated BM cells were harvested from the donor mice and injected into the tail vein of the irradiated recipients (n = 30). For analysis of the engraftment of donor BM cells, peripheral blood was obtained from the retroorbital plexus of the recipient mice using micropipettes (Drummond Scientific Co; Broomall, PA). Cells derived from donor GFP mice were detected directly on a fluorescence-activated cell sorter (FACS) [FACS Calibur; Becton Dickinson; San Jose, CA].

PH in Hypoxic Chamber

At 8 weeks after BM transplantation (BMT), the hypoxic mice were kept in a tightly sealed chamber under hypoxia (10% [O.sub.2]), which was maintained by a hypoxic air generator (TEIJIN; Tokyo, Japan) for either 4 or 8 weeks (10 mice per group). Chamber gases were monitored continuously using an [O.sub.2] analyzer (JKO-25 SII; JIKO; Tokyo, Japan). The last 10 mice were kept in the normoxic condition as a control.

Hemodynamics

Mice were anesthetized with ketamine and xylazine, and a 1.4F microtip pressure transducer (SPR-671; Millar Instruments; Houston, TX) was inserted into the RV through the jugular vein for hemodynamic measurements. RV systolic pressure (RVSP) was measured with a polygraph system (Nihon Kohden; Tokyo, Japan)

Estimation of RV Hypertrophy

The RV was dissected from the left ventricle (LV) and the septum (S), and weighed separately to determine the ratio of the RV to the LV plus S, RV/(LV + S).

Fixation and Immunohistologic Study

Mice were anesthetized with ketamine and xylazine, and the lungs were perfused from the heart with phosphate-buffered saline solution and perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline solution. Isolated lungs were immersion-fixed overnight at 4[degrees]C with rocking and subsequently were cryoprotected in sucrose solutions at 4[degrees]C. The lungs were then embedded (OCT compound; Miles Scientific; Naperville, IL) and quickly frozen-in liquid nitrogen. Cryostat sections (6 [micro]m thick) were stained with hematoxylin-eosin and elastica van Gieson stain. Other serial sections that were stained overnight at 4[degrees]C with anti-[alpha]-smooth muscle actin (SMA) [clone 1A4; Sigma Aldrich; St. Louis, MO] were used to evaluate the mobilization, distribution, and localization of GF[P.sup.+] [alpha]-SM[A.sup.+] cells. The sections were incubated for 4 h at 4[degrees]C with secondary antibodies that had been conjugated (Alexa 594; Molecular Probes; Eugene, OR). The nuclei were stained (TOTO-3; Molecular Probes). Slides were observed under a confocal laser scanning microscope (CLSM) [LSM510; Carl Zeiss; Jena, Germany) with appropriate emission filters. The GFP signal was confirmed by emission finger printing with a metaspectrometer (LSM 510 Metaspectrometer; Carl Zeiss).

Polymerase Chain Reaction

Semiquantification of mobilized GF[P.sup.+] cells was performed by polymerase chain reaction (PCR). The following primers were designed against GFP complementary DNA: 5'-primer, 5'-TGAACCGCATCGAGCTGAA GGG-3'; and 3'-primer, 5'-TCCAGCAGGACCATGTGATCGC-3'.

Statistical Analysis

Values are presented as the mean [+ or -] SEM. Statistical significance was evaluated using the unpaired Student t test for comparisons between two mean values. Multiple comparisons among more than three groups were performed using analysis of variance. A p value of < 0.05 was considered to be significant.

RESULTS

Reconstitution of BM Cells

Donor BM-derived cells engrafted successfully. FACS analysis of the peripheral blood-nucleated cells of GFP-BM-transplanted mice showed a high degree of chimerism (mean, 91 [+ or -] 2.3%) at 8 weeks after BMT (Fig 1, top, A).

[FIGURE 1 OMITTED]

Physiologic Analysis of the Hypoxia-Induced PH Mice

To confirm that prolonged exposure to hypoxia induced PH in the GFP-BM-transplanted mice, RVSP (a marker of systolic pulmonary arterial pressure) and the RV/(LV + S) ratio (a marker of RV hypertrophy) were measured. RVSP increased significantly with time in the PH mice (Fig 1, bottom left, B). There was also a significant increase in the RV/(LV + S) ratio in the PH mice, indicating RV hypertrophy (Fig 1, bottom right, C). Therefore, prolonged exposure to hypoxia induced PH with RV hypertrophy in GFP chimera mice.

Pathologic Changes in Pulmonary Arteries of the Hypoxia-Induced PH Mice

We then investigated the pathologic changes provoked by hypoxia-induced PH in the BMT mice by histologic analysis. There was marked vascular remodeling including medial hypertrophy and adventitial proliferation in both the distal pulmonary artery (Fig 2, top right, E, and upper middle, F) and the proximal pulmonary artery (Fig 2, lower middle right, G, and bottom right, H) of the PH mice compared to control mice (Fig 2, top left, A, upper middle left, B, lower middle left, C, and bottom left, D). The exposure of these animals to hypoxia led to extensive vascular remodeling of the pulmonary arteries, which was characterized by medial hypertrophy and adventitial proliferation, leading to PH.

[FIGURE 2 OMITTED]

Mobilization of BM-Derived GF[P.sup.+] Cells to the Hypoxia-Induced PH Lung

Sections from the lungs of GFP-chimeric mice in the control and the PH groups were observed by CLSM to evaluate the mobilization of BM-derived cells. In the lungs of the control mice (Fig 3, top left, A) there were a few scattered GF[P.sup.+] cells, but there was extensive infiltration of GF[P.sup.+] cells into the lungs of PH mice (Fig 3, middle left, C). Under high magnification, GF[P.sup.+] cells were found to concentrate in the pulmonary arteries, including the adventitia, in the PH mice (Fig 3, middle right, D), but not in control mice (Fig 3, top right, B). Moreover, the GF[P.sup.+] cells that were localized in the pulmonary artery wall frequently coexpressed [alpha]-SMA (Fig 3, middle right, D, bottom left, E, and bottom right, F), and were also densely clustered in the areas of expanding adventitia (Fig 3, bottom left, E). Together, these results show that BM-derived GF[P.sup.+] cells were mobilized to the pulmonary arteries in hypoxia-induced PH mice, where they expressed an SMC or myofibroblast phenotype and contributed to pulmonary vascular remodeling. It may also represent a reaction to injury associated with the presence of PH.

[FIGURE 3 OMITTED]

Contribution of BM-Derived Cells to Hypoxia-Induced Pulmonary Arterial Remodeling

To evaluate whether these mobilized BM-derived cells contribute to vascular remodeling in the PH mice, we quantified the number of GF[P.sup.+] cells that coexpressed a-SMA (GF[P.sup.+] [alpha]-SM[A.sup.+]) in the lung tissues of control and PH mice. There was a significant increase in numbers of GF[P.sup.+] [alpha]-SM[A.sup.+] cells at the pulmonary arterial wall, including the adventitia, in PH mice. Moreover, the number of mobilized GF[P.sup.+] [alpha]-SM[A.sup.+] cells increased with time (Fig 4, top, A), parallel with the progression of PH (Fig 1, middle, B, and bottom, C). Semiquantitative PCR analysis also demonstrated an increase in GFP gene expression in the lungs of PH mice (Fig 4B). Absorbance frequency analysis with a metaspectrometer (LSM 510 Metaspectrometer; Carl Zeiss) confirmed that the green signals were not due to a nonspecific background (Fig 4, bottom, C). Taken together, these results provide strong evidence that BM-derived cells, which were mobilized to the pulmonary arteries, contributed to pulmonary vascular remodeling in hypoxia-induced PH mice.

[FIGURE 4 OMITTED]

DISCUSSION

The present study examined the hypothesis that BM-derived cells are mobilized to remodeled pulmonary arteries of hypoxia-induced PH mice. We have shown that (1) hypoxia induced PH in GFP-BM chimera mice, (2) BM-derived GF[P.sup.+] cells were mobilized to the pulmonary artery walls of these hypoxic animals, and (3) a large proportion of these cells that were mobilized to remodeled pulmonary arteries, including the adventitia, expressed [alpha]-SMA, which is a marker of myofibroblasts and SMCs. Myofibroblasts play key roles in tissue remodeling, wound healing, and various fibrotic disorders, (16,17) and have been proposed to also be involved in the pathophysiology of vascular remodeling. (6,18,19) A recent study (8) showed that hypoxia induced the differentiation of pulmonary artery adventitial cells into myofibroblasts in vitro. Our present results strongly suggest that BM-derived cells that are mobilized to the pulmonary artery wall during hypoxia-induced remodeling can differentiate into myofibroblasts. These findings may indicate a reaction to injury that is associated with the presence of PH. BM-derived cells can also differentiate into SMCs in vascular disease, (9,11,20) and it is possible that the [alpha]-SM[A.sup.+] BM-derived cells that we observed in the wall may alternatively have an SMC phenotype.

It has generally been accepted that differentiation is the major mechanism by which stem cells acquire a vascular cell phenotype. (11,21,22) However, some studies (23-25) have suggested that cell fusion may mediate this process. Further studies are needed to clarify this mechanism. It will also be important in future studies to determine which BM cells are mobilized to remodeled pulmonary arteries to acquire an [alpha]-SM[A.sup.+] phenotype. There is evidence that both hematopoietie and mesenchymal stem cells can differentiate into SMCs. (9,11,26) To clarify this question, we are currently pursuing the same study using single-cell BMT mice.

CONCLUSION

BM-derived cells are mobilized to remodeled pulmonary arteries in hypoxia-induced PH mice, where they differentiate and contribute to pulmonary vascular remodeling.

* From the Cardiopulmonary Division (Drs. Hayashida, Fujita, Ogawa, and Fukuda, and Mrs. Miyake), Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan; and the Division of Hematology and Oncology (Drs. Kawada and Ando), Tokai University School of Medicine, Kanagawa, Tokyo, Japan.

Kentaro Hayashida and Jun Fujita contributed equally to this study.

Support was provided by grants from the Ministry of Education, Science and Culture, Japan, and Health Science Research for Advanced Medical Technology from the Ministry of Welfare, Japan.

REFERENCES

(1) Archer S, Rich S. Primary pulmonary hypertension: a vascular biology and translational research "work in progress". Circulation 2000; 102:2781-2791

(2) Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet 2003; 361:1533-1544

(3) Stenmark KR, Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 1997; 59:89-144

(4) Voelkel NF, Tuder RM. Hypoxia-induced pulmonary vascular remodeling: a model for what human disease? J Chn Invest 2000; 106:733-738

(5) Stenmark KR, Bouchey D, Nemenoff R, et al. Hypoxia-induced pulmonary vascular remodeling: contribution of the adventitial fibroblasts. Physiol Res 2000; 49:503-517

(6) Sartore S, Chiavegato A, Faggin E, et al. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res 2001; 89:1111-1121

(7) Stenmark KR, Gerasimovskaya E, Nemenoff RA, et al. Hypoxic activation of adventitial fibroblasts: role in vascular remodeling. Chest 2002; 122(suppl):326S-334S

(8) Short M, Nemenoff RA, Zawada WM, et al. Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts. Am J Physiol Cell Physiol 2004; 286:C416-C425

(9) Shimizu K, Sugiyama S, Aikawa M, et al. Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001; 7:738-741

(10) Saiura A, Sata M, Hirata Y, et al. Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat Med 2001; 7:382-383

(11) Sata M, Saiura A, Kunisato A, et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002; 8:403-409

(12) Hashimoto N, Jin H, Liu T, et al. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004; 113:243-252

(13) Davie NJ, Crossno JT Jr, Frid MG, et al. Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells. Am J Physiol Lung Cell Mol Physiol 2004; 286:L668-L678

(14) Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atheroselerosis of vein grafts in ApoE-deficient mice. J Clin Invest 2004; 113:1258-1265

(15) Okabe M, Ikawa M, Kominami K, et al. "Green mice" as a source of ubiquitous green ceils. FEBS Lett 1997; 407:313-319

(16) Kuhn C, McDonald J. The roles of the myofibroblast in idiopathic pulmonary fibrosis: ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol 1991; 138:1257-1265

(17) Bogatkevich GS, Tourkina E, Silver RM, et al. Thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the proteolytically activated receptor-1 and a protein kinase c-dependent pathway. J Biol Chem 2001; 276:45184-45192

(18) Shi Y, O'Brien JE, Fard A, et al. Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries. Circulation 1996; 94:1655-1664

(19) Li G, Chen S-J, Oparil S, et al. Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries. Circulation 2000; 101:1362-1365

(20) Caplice NM, Bunch TJ, Stalboerger PG, et al. Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc Natl Acad Sci U S A 2003; 100:4754-4759

(21) Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107:1395-1402

(22) Bailey AS, Jiang S, Afentoulis M, et al. Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells. Blood 2004; 103:13-19

(23) Terada N, Hamazaki T, Oka M, et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002; 416:542-545

(24) Wagers AJ, Sherwood RI, Christensen JL, et al. Little evidence for developmental plasticity of adult hematopoietie stem cells. Science 2002; 297:2256-2259

(25) Ying QL, Nichols J, Evans EP, et al. Changing potency by spontaneous fusion. Nature 2002; 416:545-548

(26) Kinner B, Zaleskas JM, Spector M. Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 2002; 278:72-83

Manuscript received July 22, 2004; revision accepted November 22, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjourual. org/misc/reprints.shtml).

Correspondence to: Keiichi Fukuda, MD, FCCP, Cardiopulmonary Division, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; e-mail: kfukuda@sc.itc.keio.ac.jp

COPYRIGHT 2005 American College of Chest Physicians
COPYRIGHT 2005 Gale Group