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Ann Thorac Surg 2007;84:544-552
© 2007 The Society of Thoracic Surgeons
Division of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, P.R. China
Accepted for publication March 20, 2007.
* Address correspondence to Dr Zixiong Liu, Division of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, 200032, P.R. China (Email: pieero{at}sina.com).
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Abstract |
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Methods: Endothelial progenitor cells were obtained from cultured human peripheral blood mononuclear cells. The genetic sequence for CGRP was subcloned into cultured EPCs by human expression plasmid. Pulmonary hypertension was established in immunodeficient rats with an abdominal aorta to inferior vena cava shunt operation. The transfected EPCs were injected through the left jugular vein at 10 weeks after the shunt operation. Mean pulmonary artery pressure and total pulmonary vascular resistance were detected with right cardiac catheterization at 4 weeks. The distribution of EPCs in the lung tissue was examined with immunofluorescence technique. Histopathologic changes in the structure of the pulmonary arteries was observed with electron microscopy and subjected to computerized image analysis.
Results: The lungs of rats transplanted with CGRP-expressing EPCs demonstrated a decrease in both mean pulmonary artery pressure (17.64 ± 0.79 versus 22.08 ± 0.95 mm Hg; p = 0.018) and total pulmonary vascular resistance (1.26 ± 0.07 versus 2.45 ± 0.18 mm Hg · min/mL; p = 0.037) at 4 weeks. Immunofluorescence revealed that intravenously administered cells were incorporated into the pulmonary vasculature. Pulmonary vascular remodeling was remarkably attenuated with the administration of CGRP-expressing EPCs.
Conclusions: The transplantation of CGRP-expressing EPCs may effectively attenuate established pulmonary hypertension and exert reversal effects on pulmonary vascular remodeling. Our findings suggest that the therapy based on the combination of both CGRP transfection and EPCs may be a potentially useful strategy for the treatment of pulmonary hypertensive disorders.
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Introduction |
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Calcitonin gene-related peptide (CGRP) has a strong pulmonary vasodilator activity, and its level is reduced in the case of pulmonary hypertension. Previous studies have shown CGRP can inhibit pulmonary smooth muscle cell proliferation by binding to CGRP receptors and confer beneficial effects on pulmonary hypertension [7–9]. The purpose of this present investigation was to study the effect of intravenously administered EPCs containing CGRP gene on the attenuation of elevated mean pulmonary arterial pressure (mPAP) and pulmonary vascular remodeling induced by left-to-right shunt in rat models. To test this hypothesis we constructed plasmids including CGRP gene and subcloned them into the eukaryotic expression vector pEGFP-N1, which was then delivered into human EPCs by electroporation. Thus, we anticipated the intravenously administered CGRP gene–transduced EPCs might be incorporated into the pulmonary vasculature, repairing the impaired endothelium and reversing the vascular remodeling. This strategy may prove to be a promising way for the treatment of pulmonary hypertension in the future.
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Material and Methods |
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Isolation and Culture of Endothelial Progenitor Cells
Total peripheral blood mononuclear cells was isolated from 20 mL of peripheral blood, obtained from healthy human volunteers, by density-gradient centrifugation and suspended in M199 medium containing 20% fetal bovine serum, basic fibroblast growth factor (2 µg/L, CytoLab, Rehovot, Israel), vascular endothelial growth factor (10 µg/L, CytoLab), penicillin (100 U/mL), streptomycin (100 µg/mL), and heparin (100 U/mL); 1 x 105 mononuclear cells per well were seeded on fibronectin (Sigma Chemical Company, St. Louis, MO) -coated six-well plates and incubated in 5% CO2 at 37°C [10–12]. Four days later the medium solution was renewed, and nonadherent cells were removed. On day 7 the adherent cells were harvested and underwent cell counting under the microscope.
Endothelial Progenitor Cell Culture Assay
The harvested adherent cells on day 7 were incubated in culture medium containing 1,1'-dioctadecyl-3,3,3,'3'-tetramethylindocarbocyanine–labeled acetylated low-density lipoprotein (ac-LDL,15 µg/L; Molecular Probes, Eugene, OR) for 24 hours and then fixed in 4% paraformaldehyde for 10 minutes. After washing with phosphate-buffered saline solution, the cells were left reacting with fluorescein isothiocyanate–labeled lectin from Ulex europaeus agglutinin (UEA-1, 10 µg/L; Sigma) for 1 hour. The samples were observed under an inverted fluorescent microscope. Cells exhibiting red fluorescence were defined as ac-LDL positive, cells exhibiting green fluorescence were defined as UEA-1 positive, and those double-positive cells were identified as EPCs. Most of the adherent cells (85%) were ac-LDL and UEA-1 positive. Adherent cells on day 7 of culture were incubated with mouse monoclonal antibodies against human vascular endothelial growth factor receptor-2, CD34, and CD133 (Dako, Hamburg, Germany) for 1 hour at 4°C. Then the cells were incubated with fluorescein isothiocyanate–conjugated goat anti-mouse antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for 45 minutes. After washing again, cells were fixed with 4% paraformaldehyde and analyzed by fluorescence-activated cell sorting (FACS; Becton, Dickinson and Company, Franklin Lakes, NJ). Isotype-identical antibodies served as the control [13–16].
Construction of Calcitonin Gene-Related Peptide Gene Expression Vector
A pair of primers for the CGRP gene open reading frame was designed according to the whole genetic sequence of CGRP. Restriction endonuclease EcoRI and BamHI sites were introduced into the 5' end of the upstream and downstream primer sequences, respectively. Primer sequences were 5'-GAATTCTCCTGCAACACCGCCACCTG-3' and 5'-GGATCCGGTGGGCACAAAGTTGTCCT-3'. The plasmid vector encoding the human CGRP gene (pGEM-T-CGRP, generated by Fudan University Biotech Center, Shanghai, China) was used as a template. Polymerase chain reactions were cycled 35 times at 94°C (denaturation) for 1 minute, 60°C (annealing) for 1 minute, and 72°C (extension) for 1 minute. Samples were incubated at 72°C for an additional 10 minutes after the last cycle was completed. The polymerase chain reaction products were loaded on 1% agarose gel and electrophoresed. Both polymerase chain reaction products and the plasmid vector pEGFP-N1 were digested with restriction endonucleases EcoRI and BamHI. The CGRP gene was cloned into the multiple cloning sites of pEGFP-N1 right between the human cytomegalovirus immediate early promoter and the green fluorescence protein coding sequences. After purification the resulting DNA fragments were ligated and introduced into bacteria (E. coli DH5
) by heat-shock transformation. Transformants were selected in culture medium supplemented with kanamycin (100 µg/mL); whole colonies were chosen to undergo plasmid amplification, extraction, and sequence analysis.
Gene Transfer Into Cultured Endothelial Progenitor Cells
Endothelial progenitor cells were transfected with the reconstructed CGRP gene–encoding plasmid vector through electroporation. After transfection, cells were replated on six-well plates with 1 mL of 20% fetal bovine serum per well and cultured for 24 to 48 hours. Cell transfection was examined through the observation of green fluorescence with the fluorescent microscope. The transfected cells were trypsinized (0.25% trypsin), washed, and resuspended, and then divided into aliquots of 2 x 106 cells/mL intended for injection.
Cell Delivery Into Rats
Cell transplantation was performed at 10 weeks after shunt operation. Cells (1 x 106 EPCs or 1 x 106 CGRP-expressing EPCs, 500 µL each) were administered intravenously through the left jugular vein to the shunt + EPCs group and the shunt + CGRP-EPCs group, respectively, whereas the shunt and control groups received intravenous administration of 500 µL of culture medium. After injection all the animals continued to be bred in the pathogen-free environment for a subsequent period of 4 weeks. Hemodynamic studies were performed at the end of 4 weeks.
Measurement of Pulmonary and Systemic Responses
The anesthetized animals were strapped in a supine position, and a specially designed polyethylene single-lumen catheter with a curved tip was inserted through the right jugular vein to course through the right heart, into the pulmonary artery. Pulmonary artery pressure was measured and recorded with a pressure transducer attached to a polygraph. Cardiac output was determined with the thermodilution technique by injection of a known volume of 0.9% saline solution at 23°C into the right atrium. A thermistor microprobe was inserted into the right carotid artery and advanced to the aortic arch to measure the changes in aortic blood temperature. The temperature dilution curve and systemic arterial pressure were recorded.
Assay for Lung and Plasma Calcitonin Gene-Related Peptide Levels
Rats were sacrificed, and the left lobe of the lung was excised and quick-frozen in liquid nitrogen. After lung tissues were homogenized, the homogenates were boiled in saline solution for 15 minutes to inactivate intrinsic proteases and then centrifuged (24°C, 1,500 g for 30 minutes) with supernatants collected. Boiling the lung pellet was repeated in 0.5 mol/L acetic acid for 15 minutes, followed by centrifugation. The combined supernatants were lyophilized until assay. Radioimmunoassay was performed on lung extracts, culture medium, and plasma with a radioimmunoassay kit (East Asia Immune Tech, Beijing, China).
Incorporation of Endothelial Progenitor Cells Into the Pulmonary Vasculature
CD31 is a common marker on native endothelial cells in the pulmonary vasculature, so immunofluorescence staining for rat CD31 was performed on frozen sections with mouse anti-rat CD31 monoclonal antibody and rhodamine-B isothiocyanate–conjugated anti-mouse immunoglobulin G antibody (Sigma), which makes the rat pulmonary vasculature appear red on the background in contrast to the human green fluorescence protein–tagged EPCs that appear green under the fluorescence microscope.
Morphometric Analysis of Pulmonary Arteries
The animals were sacrificed as described previously, and the right lobe of lung tissue was obtained, fixed with 10% paraformaldehyde, embedded in paraffin, cut into sections approximately 4 µm thick, and stained with hematoxylin and eosin. Three cross sections were randomly chosen from each rat, and pulmonary arteries with diameters ranging from 100 to 150 µm underwent computerized image analysis to determine the ratio of vessel wall area to total area, the ratio of vessel lumen area to total area, and the wall thickness to assess the degree of pulmonary vascular remodeling. Another piece of lung tissue (10 mm x 10 mm x 15 mm) was obtained, and three small pieces (1 mm3 each) were cut from it, then immediately fixed in 2.5% glutaraldehyde. After a series of gradient dehydration steps, embedding, solidification, and staining, the specimens were observed with a transmission electron microscope to track the changes in pulmonary artery ultrastructure.
Survival Analysis
In a separate study 24 rats received intravenous injection of 1 x 106 EPCs (n = 8), 1 x 106 CGRP-EPCs (n = 8), or culture medium (n = 8) 10 weeks after the shunt operation. Survival was observed from 10 weeks after the shunt operation when cell injection was performed until the death of the rats or 90 days after cell transplantation.
Statistical Analysis
Data are presented as mean ± standard deviation. Differences among groups were analyzed by one-way analysis of variance and further analyzed by Student-Newman-Keuls test for multiple comparisons among groups. Significance of differences for survival data was determined using the Kaplan–Meier analysis. Probability values less than 0.05 were considered to be statistically significant. All statistical analysis was finished with SPSS 13.0 statistical software packet (SPSS, Chicago, IL).
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Results |
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Comment |
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Endothelial progenitor cells have recently been identified as a circulating cell population in the peripheral blood [17]. Circulating EPCs differ from the circulating endothelial cells that are randomly detached from the vessel walls. When incubated in vitro with appropriate medium in the presence of specific growth factors, EPCs have a high proliferating potential and within 10 days produce colonies of cells expressing endothelial cell markers. Previous studies have established that EPCs express the cell surface markers CD34, CD133, and vascular endothelial growth factor receptor-2 and also demonstrate the capacity of taking up ac-LDL and binding to UEA-1, which are characteristic features of endothelial lineage cells. Studies on the more immature hematopoietic stem cell marker CD133 demonstrated that CD133-positive cells that concomitantly express the vascular endothelial growth factor receptor-2 can differentiate to endothelial cells in vitro. CD133, also known as prominin or AC133, is a highly conserved antigen that is expressed on hematopoietic stem cells but is absent on mature endothelial cells and monocyte cells. Overall, there is consensus that ac-LDL and UEA-1 double-positive cells coexpressing CD133 and vascular endothelial growth factor receptor-2 represent a population with an endothelium-oriented differentiating capacity.
The application of EPCs as a tool for cell therapy in diseases involving defects of the local vascular structure was envisioned as soon as these cells were discovered. In many experiments performed in different models, it was proved that these cells were potent in the repairing of local vascular tissues, and their specific tropism for damaged tissues was encouraging [18–20]. According to a recent report, intravenously administered EPCs will home in on ischemic myocardium and congregate there after myocardial infarction, indicating the EPCs are capable of sensing the impaired tissues that need to be restructured. Endothelial progenitor cells can also act as an effective therapeutic gene vehicle because they are readily cultured and transfected in vitro, thus avoiding the fact of low transfection rate related to in vivo gene transfection. Moreover, using EPCs as a gene vehicle will protect the target gene from degradation in the circulation. These traits give EPCs a special superiority in finding out the target area to perform therapeutic functions. In this study we cloned the CGRP gene into EPCs to test the feasibility and efficacy of progenitor cell–based gene therapy for the treatment of pulmonary hypertension.
Calcitonin gene-related peptide is a 37-amino acid endogenous peptide formed as an alternative splice product of the calcitonin gene and is a potent vasodilator and vascular smooth muscle proliferation inhibitor. The binding of CGRP on cell surface receptors activates a signaling pathway mediated by cyclic nucleotides that results in inhibition of cell proliferation. Previous studies have shown the loss of CGRP will inevitably result in the proliferation of vascular smooth muscle cells and pulmonary vascular remodeling as in the case of pulmonary hypertension. Calcitonin gene-related peptide and nitric oxide are by far the two most potent vasodilators that interest researchers, and both agents have been shown to alleviate the symptoms of pulmonary hypertension [21–24]. Calcitonin gene-related peptide can raise the intracellular cyclic adenosine monophosphate levels, which in turn stimulate the nitric oxide production by endothelial cells. Thus, the importance of CGRP in a protective effect for pulmonary hypertension deserves further investigation.
The results of the present study demonstrate that transplantation of CGRP-expressing EPCs attenuates increases in pulmonary artery pressure. Pulmonary vascular morphologic examination also showed that pulmonary vascular remodeling was remarkably inhibited as the vascular medial wall thickness was reduced. Under normal conditions, vascular smooth muscle cells in the medial layer are in a stable status and do not proliferate. Injury to the vessel wall endothelial cells such as in the case of high-volume perfusion increases smooth muscle cell responsiveness to growth factors, resulting in the loss of inhibition of proliferation and vascular remodeling. The ultrastructural observations demonstrated the degeneration and necrosis of the endothelial cells as well as the transformation of smooth muscle cells from a contractile phenotype to a synthetic phenotype. Thus, it is reasonable to administer viable EPCs to settle on the injured vascular inner surface and differentiate into normal endothelial cells to redress this problem. Through the observation of green fluorescence distributed in lung tissues, it can be inferred that the CGRP gene-modulated EPCs were incorporated into the pulmonary vasculature where CGRP could readily play its role in regulating pulmonary vascular tone and smooth muscle proliferation without causing overall systemic hemodynamic disturbance. However, it should be noted that the distribution of EPCs was not uniform throughout the lungs. In areas with fewer transfected EPCs, there was still obvious improvement in lung pathologic changes, which may be related to the paracrine effect of CGRP.
In vivo studies have shown that pulmonary CGRP levels are associated with protection (higher levels) and exacerbation (lower levels) of vascular smooth muscle remodeling [25, 26]. In the present study lung CGRP levels were found to be slightly increased after the shunt operation, probably related to the protective mechanism of the body in response to high-volume lung perfusion, but not increased enough to counteract the ongoing pathologic changes. Through the transplantation of CGRP gene-transduced EPCs, higher levels of CGRP were detected in lung tissues accompanied with decreases in mPAP and total pulmonary vascular resistance, thus indicating that CGRP was effectively produced and played an important role in alleviating the pulmonary hypertension. In animals receiving only EPC transplantation, a moderate decrease in total pulmonary vascular resistance was also observed, which may be related to the ability of EPCs in restoring normal endothelial function through repairing impaired tissue, whereas the transplantation of CGRP-expressing EPCs manifested its effects on lowering both mPAP and total pulmonary vascular resistance. Plasma CGRP levels were decreased in all the animals after the shunt operation, and there was no difference between animals receiving only the shunt operation and those receiving EPC or CGRP-EPC transplantation. No major upheavals in systemic hemodynamic indices such as mean arterial blood pressure, heart rate, cardiac output, or systemic vascular resistance were detected. The study showed that although the majority of intravenously administered EPCs home in on the injured pulmonary vasculature, there was still incorporation of EPCs, although fewer, into cardiac tissue that may account for another mechanism of stabilized hemodynamics in volume-overload pulmonary hypertension.
The current perioperative treatment of severe pulmonary hypertension with traditional agents such as nitrovasodilators, ß-adrenergic agonists, and phosphodiesterase inhibitors is limited by the risk of hypotension as a result of their systemic vasodilatory effects [27, 28]. Patients with preexisting pulmonary hypertension seem to be at an increased risk because higher doses of these agents are required [29]. Recent clinical trials have demonstrated the efficacy of inhalation of nitric oxide in patients during congenital heart disease repair [30, 31]. However, the application of nitric oxide inhalation is limited as it requires delivery through a closed system and patients need an endotracheal tube or a sealed mask, which complicates the clinical management and often produces unfavorable side effects. In addition, it is often the case that on withdrawal of nitric oxide, rebound pulmonary hypertension would occur. It would be of considerable therapeutic benefit to develop a method for delivery of vasodilator agents directly to the pulmonary vascular bed.
The gene delivery system tested in this study has great advantages over conventional gene therapy: nonviral, less invasive, and highly efficient cell incorporation into the pulmonary vasculature without major systemic hemodynamic disturbances. These findings may have important implications with respect to better understanding the mechanisms underlying the development of pulmonary hypertension, and identifying potential therapeutic strategies to prevent development, retard progression, and even induce regression of pulmonary vascular disease.
Previous studies showed adenoviral transfection of CGRP alone yielded improved hemodynamics in hypoxia-induced pulmonary hypertension. It is worth pointing out that there is no artificially synthesized CGRP available, plasmid transfection alone cannot produce a satisfactory transfection rate with an adenoviral method, and this study aimed at a virus-free approach, so no control group of rats receiving only CGRP was set up.
In summary, this study generated preliminary positive results in regard to the progenitor cell-based CGRP gene delivery in the treatment of pulmonary hypertension. Further studies, including taking into consideration the specific determination of shunt fraction, are still needed, which would be helpful in presenting a more complete picture.
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This article has been cited by other articles:
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  D. J. Weiss, J. K. Kolls, L. A. Ortiz, A. Panoskaltsis-Mortari, and D. J. Prockop Stem Cells and Cell Therapies in Lung Biology and Lung Diseases Proceedings of the ATS, July 15, 2008; 5(5): 637 - 667. [Full Text] [PDF]
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 N. K. Kapur Invited commentary Ann. Thorac. Surg., August 1, 2007; 84(2): 552 - 552. [Full Text] [PDF]
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