Autologous Endothelail Progenitor Cells Transplantation For The Therapy Of Primary Pulmonary Hypertension: An Empirical Study

Background:Primary pulmonary hypertension (PPH) is a progressive disease of unknownetiology manifested by increased pulmonary vascular resistance and progressive rightheart failure. The pulmonary arteries in PPH are characterized by intimal fibrosis,medial hypertrophy, adventitial proliferation, and obliteration of small arteries. PPHresponds poorly to conventional therapeutic agents and most of the patients diewithin 2 to 3 years from the diagnosis. Although new classes of drugs such asprostanoids, endothelin receptor antagonists and type 5 hosphodiesterase inhibitors1has been shown to exert favourable effects on PPH patient's haemodynamics,exercise capacity and survival in patients with severe PPH, the long-term prognosisof the disease is still poor. Therefore, it is of paramount importance to develop newtherapeutic strategies for this devastating disease. Our understanding of thepathogenetical and pathobiologic mechanisms underlying PPH has progressedrapidly over the past few years. Current evidence strongly suggests a central role forendothelial dysfunction and cell loss in the initiation and progression of PPH. It isrecognized that a number of stimuli, including shear stress from increased pulmonaryblood flow, viral infection (HIV), and alveolar hypoxia, may potentially injure endothelium in genetically predisposed individuals. Injury to the endothelium leads to apoptosis of the usually quiescent cells, destabilization of the pulmonary vascular intima, and disordered endothelial cell proliferation. Pulmonary endothelial dysfunction results in an altered production of vasodilators, such as nitric oxide and prostacyclin, and over expression of vasoconstrictors, such as thromboxane A₂ and endothelin-1, which lead to pulmonary vasoconstriction. The loss of endothelial barrier integrity permit the extravasation of factors that initiate growth signals to medial smooth muscle cells. In addition, vascular smooth muscle and adventitia may appear adaptive hypertrophy in response to the increase of luminal pressure. Endothelial dysfunction increases the production of various pro-thrombotic and decreases the production of anti-thrombotic substances, resulting in a hypercoagulable state. Thrombosis narrows the pulmonary vessel lumen, which may help propagate the changes of PPH. These evidences suggest endothelial dysfunction play an integral role in mediating the structural changes in the pulmonary vasculature.Endothelial progenitor cells(EPCs) are a subset of pluripotent "stem cells" derived from the bone marrow. Accumulating evidence suggests that EPCs plays an important role in postnatal neovascularization of adult ischaemic tissues. Further studies have demonstrated that release of endothelial progenitor cells from the marrow into the peripheral blood occurs in response to ischemia, trauma, exogenous cytokine and drug to reach a neoangiogenic site there they promote neovascularization and improve endothelial function. EPCs provide a circulating pool of cells that could form a cellular patch at the site of denuding injury or serve as a cellular reservoir to replace dysfunctional endothelium. Autologous EPCs transplantation may be a feasible adjunctive therapeutic option for PPH.The aim of this study are:l) to establish canine pulmonary hypertension ( PH)model. 2)to investigate quantitive and functional change of circulating EPCs in PH dogs. 3)to transplate autologous EPCs into canine pulmonary circulation. 4) to investigate the mechanisms of EPCs transplantation as a therapeutic method for PH by observing hemodynamic and histological changes of pulmonary circulation and provide empirical and theoretical foundation for the cell therapy of PPH.Methods:Animals and treatment14 male beagles were labeled and divided into three groups: T group(6 dogs for transplantation), C group((6 dogs as control) and S group(2 dogs as sham).Induction of PH in dogsAnimals were anesthetized with pentobarbital (30mg/kg body weight, peritoneal injection) and breathe spontaneously. After normal hemodynamic measurements were obtained, T and C Groups were administered via the right ventricle with 3mg/kg of dehydromonocrotaline(DHMC) to induce PH and S group were injected with the same volume of dimethylformamide. Six weeks later, hemodynamic changes were evaluated.Hemodynamic measurementsA 5F Swan-Ganz catheter was advanced through the right jugular vein into the right atrium, the right ventricle, and the pulmonary artery to measure pulmonary hemodynamics including central venous pressure(CVP). right ventricular pressure(RVP), pulmonary arterial systolic pressure(PASP), mean pulmonary arterial pressure(mPAP) and pulmonary capillary wedge pressure(PCWP). Systemic arterial pressure was obtained through a catheter inserted into the left femoral artery. Cardiac output was measured in triplicate by the thermodilution method. Total pulmonary vascular resistance(TPVR) was calculated by dividing mean pulmonary arterial pressure by cardiac output.Blood gas analysis and endothelinBlood gas analysis and endothelin examination were progressed at baseline, the end of the 6^th week and the end of the 10^th week after DHMC injection.Quantitive and functional examination of EPCs20ml blood was obtained via right jugular vein after pulmonary hemodynamic examination at the baseline and 6 weeks after DHMC injection from 12 dogs(T and C group). Mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque and cultured in vitro. UEA-I and DiLDL positive cells were counted through inverse fluorescent microscope. KDR and AC 133 positive cells were analyzed by fluorescence-activated cell sorting. Adheral, migratory and vessel formative capability were examed.EPCs transplantation60ml blood was obtained through right femoral vein 35days after DHMC injection. EPCs were expanded and phaenotype was detected by flow cytometer. To track transplanted cells, EPCs were labeled by Dil staining and Feridex in vitro labeling(2 dogs respectively). Six weeks after DHMC injection, T group received EPCs transplantation, C and S groups were injected with the same volume of culture solution.MRITwo dogs transplanted with EPCs labelled by Feridex were scaned before and after transplantation by MRI with SE - T2W I, FSE - T2W I and GRE - T2W I sequence.Histological evaluationAnimals were sacrificed at the end of the 10^th week and heart and lung were taken out. Right lung tissues underwent 5μm frozen section after nitrogen fixation and examed under fluorescence microscope to identify EPCs. The other lung tissues were stained for histomorphologyical study. The vessels in the lung tissues were counted under a light microscope. The thickness percentage of pulmonary arteriole media and right heart index were counted.Results:PH model11 dogs survived after DHMC injection, and one dogs from C group died at the second day. Six weeks later, hemodynamics results showed that mPAP incrased from 11.4±1.7 to 19.4±1.5 mmHg(t= 16.455, P<0.001), TPVR increased from 3.2±0.7 mmHg·min·m²/L to 5.1±0.8 mmHg·min·m²/L(t=4.969, P=0.001). Hear rate, central vein pressure, systemic systolic pressure, right ventricular pressure and cardiac index did not show statistic significance. Blood gas analysis results had no statistic significance and plasma endothelin levels rise from 1.37±0.03 pg/ml to 1.63±0.04 pg/ml(t=3.010, P=0.013).Characteristics of EPCsAt first week, about 25% isolated PBMCs plated on fibronectin attached. All attached and nonattached cells present round morphological appearance and became spindle shaped like that of mature endothelial cells at the second week. At the end of the third week, they looked typically like cobble. At 7^th day attached cells expressed KDR:78.0±7.8%, CD34:28.7±6.9% and AC133:17.1±8.1% and most attched cells showed the ability to intake UEA-I and DiLDL under fluorescence microscope.EPCs quantity and function after PH formationAfter PH formation, the number of AC133+KDR+ cells declined from 633.8±39.3 cells/ml to 210.4±26.7 cells/ml(t=32.758,P<0.001) and both UEA-I and DiLDL staining positive cells declined from 42.2±5.6 cells /×200f ield to 23.3±6.1 cells/×200 field (t=10.679,P<0.001). Attached cells quantity declined from 32.7±8.2 cells/×200 field to 28.0±5.7 cells/×200 field (t=2.891, P=0.016) . The number of cells migrating to lower chamber also declined from 12.6 +++ 4.7 cells/xxx 200 field to 9.4±3.4 cells/×200 field ( t=2.292, P=0.045 ) . Blood vessel formation test in vitro showed the number of vessels was 11.7±3.0/×200 field after PH formation while it was 20.8±2.8/×200 field at baseline(t=13.108, P<0.001). The quality and complication of vessels formed by EPCs after PH formation was inferior to that at baseline.Hemodynamics after EPCs transplantationFour weeks after EPCs transplantation, mPAP of C group continue to increase from 19.4±1.95 mmHg to 28.6±3.05mmHg (t=15.778, P<0.001 ) , while mPAP of T group decline from 19.3±2.25 mmHg to 16.2±5.78mmHg ( t=1.172, P=0.294) . mPAP of T group was significantly lower than C group at the end of 10^th week (t=4.312, P=0.002) . TPVR of T group was also significantly lower than C group ( 3.99±1.07 vs 7.75±1.29, t=5.292, P<0.001) .Endothelin levels after EPCs transplantationPlasma endothelin levelof C group went on rising from 0.47±0.04 pg/ml at 6^th week to 0.53±0.03pg/ml at 10^th week (t=2.851, P=0.046) while T group had no significance ( 0.42±0.05 pg/ml vs 0.41±0.09 pg/ml, t=0.357, P=0.736 ) . However, plasma endothelin level of T group was lowe than that of C group at the end of 10^th week (t=2.885, P=0.018) .Histological evaluationThe appearance of lungs of normal dogs present pink and the color was uniform while that of dogs administered with DHMC prsetent dark red with pale even black strip and flake region. Pulmonary arteriole media became thick and lumina became narrow. The percentage of media thickness of S group was (8.4±3.2 ) % ,C group ( 17.2±5.3 ) %, and T group ( 14.2±5.2 ) %, there were statistic differences among the three groups (F=8.952, P=0.001 ) ; although T group was lower than C group in numerical value, there was no statistic difference between the two groups. The vessels in the lung tissues of T group under a light microscope (×40 magnification) were 305±35/20 field while 284±34/20 field of C group, showed no statistic significance (t=0.828, P=0.429) . There were no statistic differences among the three groups in right ventricular hypertrophy index ( F=1.369, P=0.298) .EPCs tracingFour weeks after EPCs transplantation, frozen sections of animals' lungs did not show red fluorescent cells and MRI scan did not find abnormal low signal alteration in lungs and other organs.Conclusions:1. DHMC induced canine model showed increased pulmonary arterial pressure and total pulmonary vascular resistence, thickening of pulmonary arteriole media and increased endothelin level, which confirmed the establishment of chronic canine pulmonary hypertensive model.2. PH dogs had less quantity of EPCs isolated from peripheral blood and cultured in vitro, impacted ability of adherence and migration, and subsided capability of vessel formation.3. Autologous EPCs transplantation could improve canine pulmonary haemodynamics.4. The therapeutic function of EPCs might due to paracrine which improving endothelia function by promoting proliferation, migaration and differentiation of vascular endothelial cell.