Transplantation Of Prepro-Calcitonin Gene-Related Peptide Expressing Endothelial Progenitor Cells Attenuates Left To Right Shunt Induced Pulmonary Hypertension In Rats

Vascular remodeling resulting from SMC proliferation and migration characterize pulmonary hypertension pathegensis. It is known that SMC is subject to the regulation of cytokines from endothelium. [1-4] . Recently EPCs have been discovered in human peripheral blood, capable of migrating to sites of injured endothelium and differentiating into mature functional endothelial cells[5,6] . This indicates EPCs may play an important role in the treatment of pulmonary hypertension since they can not only repair damaged lung tissue but also serve as a vehicle for the specific genes responsible for reversing pulmonary vascular remodeling. 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 SMC proliferation by binding to CGRP receptors on vascular SMCs and confer beneficial effects on pulmonary hypertension. Calcitonin gene-related peptide (CGRP) is an potent smooth muscle cells (SMC) proliferation inhibitor and vasodilator. It is now believed CGRP plays an important role in maintaining a low pulmonary vascular resistance (PVR).We evaluated the therapeutic effect of intravenously administered CGRP gene transduced endothelial progenitor cells (EPC) on left-to-right shunt Induced pulmonary hypertension in rats.MethodEPCs were obtained from peripheral blood mononuclear cells isolated from 20ml peripheral blood of healthy human volunteers by density gradient centrifugation and cultured in medium containing basic fibroblast growth factor, vascular endothelial growth factor. The harvested adherent cells on day 7 were tested for immunoflourescence with DiI-acLDL and UEA-1, CD34 and CD133 were assayed with FACS. CGRP gene was subcloned into the multiple cloning sites of pEGFP-N1 right between the immediate early promoter of CMV and the EGFP coding sequences. Transformants were selected and undergo plasmid amplification and extraction. EPCs were transfected with human pEGFP-CGRP plasmid vector generated using electroporation. 1×10~6 EPCs, 1×10~6 CGRP-expressing EPCs (500uL each) was administered intravenously via the left jugular vein to Shunt±EPCs group, shunt±EPCs-CGRP group respectively, while shunt group and control group received intravenous administration of 500 uL of culture medium. Pulmonary hypertension was established in immunodeficient rats with abdominal aorta-inferior vena cava shunt operation. Mean pulmonary artery pressure (mPAP) and PVR were detected with right cardiac catheter in 4 weeks. Radioimmunoassay for CGRP was performed on lung extracts, culture medium and plasma with a radioimmunoassay kit. The distribution of EPCs in lung tissue was examinated with immunofluorescence technique with mouse anti-rat CD31 monoclonal antibody and RITC conjugated anti-mouse IgG antibody. Lung CGRP mRNA levels were determined with RT-PCR Assay. Cross sections of lung tissue were randomly chosen from each rat and pulmonary arteries with diameters ranging from100．m to 150．m underwent computerized image analysis to determine vessel wall area to total area ratio(WA%),vessel lumen area to total area ratio(LA%) and wall thickness(WT) in order to assess the degree of pulmonary vascular remodeling. The ultrastructure of pulmonary arteries were observed with transmission electronmicroscope. In a separate study 24 rats received intravenous injection of 1×10~6 EPCs (n=8) , 1×10~6 CGRP-EPCs (n=8) , or culture medium (n=8) 10 weeks after shunt operation. Survival was observed from the date when injection of cells were performed till the death of the rats or 90 days after cell transplantation.Result1．Forty eight hours after culture EPCs from human peripheral blood became adherent. On day 4 of culture, the cells took the shape of s pindle or cobblestone, and appeared in clusters resembling the structure of a blood island. On day 7 the number of spindle-shaped cells increased. Over 85 percent of the adherent cells demonstrated double positive for ac-LDL and UEA-1 staining in accordance with endothelial cell function. Flow cytometry indicated most adherent cell were positive for specific cell surface marker( CD34 : 77．2 %±10．9%, AC133 : 27．1%±9．1%). 2．Twenty-four hours after gene transfer into EPCs, CGRP was detected in the cell culture medium. On day 5 the CGRP concentration reached its peak, while the period of CGRP overproduction lasted for more than 3 weeks. Concentrations of CGRP in lung increased in rats receiving shunt operation compared with control group. CGRP concentrations were significantly increased in lungs of rats with CGRP-EPC transplantation compared with those from shunt group or shunt±EPCs group. (P<0．05) . Plasma concentrations of CGRP were significantly reduced in all three groups receiving shunt operation compared with control rats. No elevated plasma CGRP values were recorded in EPC-CGRP group compared with shunt group or EPC group.3．Twenty-four hours after gene transfer into EPCs, CGRP was detected in the cell culture medium. On day 5 the CGRP concentration reached its peak, while the period of CGRP overproduction lasted for more than 3 weeks. Concentrations of CGRP in lung increased in rats receiving shunt operation compared with control group. CGRP concentrations were significantly increased in lungs of rats with CGRP-EPC transplantation compared with those from shunt group or shunt±EPCs group. (P<0．05) . Plasma concentrations of CGRP were significantly reduced in all three groups receiving shunt operation compared with control rats. No elevated plasma CGRP values were recorded in EPC-CGRP group compared with shunt group or EPC group.4．Four weeks after ex vivo transfer of EPCs or EPC-CGRP, CGRP and .-actin mRNA expression was determined in lung tissue from all groups of rats. The mRNA for CGRP was expressed in lung tissue from all four groups of animals (90bp). CGRP mRNA levels were analyzed by densitometry and normalized by dividing each integrated density value by that of the .-actin (350bp).The expression of CGRP was significantly higher in rats treated with EPC-CGRP compared with shunt group or shunt±EPCs group(P<0．05) .5．24 hours after gene tranduction into EPCs green fluorescence was observed within the cells. Four weeks after cell delivery CGRP-EPCs were found incorporated into tissues of lung in rats and became part of the structure of pulmonary vascular beds as demonstrated by the observation of green fluorescence distributed across lung tissues. No obvious distribution of green fluorescence in tissues other than lungs was detected.6．Lung sections were examined histologically to determine the influence of CGRP-EPCs on pulmonary vascular remodeling in response to high volume pulmonary blood flow. When compared to shunt group, rats treated with either EPCs or CGRP-EPCs showed a significantly greater value of LA% and a lesser value of WA% (P<0．05) . Quantitative analysis demonstrated a significant increase in pulmonary artery wall thickness after shunt surgery, but this change was remarkably attenuated in the CGRP-EPC treated group. Notably rats treated with CGRP-EPCs had significantly greater improvements in vascular structure than those treated only with EPCs(P<0．05) .7．The changes in ultrastructure of pulmonary arteries in shunt group included that endothelial cells became swollen and large in size or peeled off, endo-elastic membrane dissolved, extracellular matrix and collagen deposited in pulmonary walls. Endothelial cell regeneration and degeneration were found coexistent in shunt±EPC group, while CGRP-EPC treated rats showed steady connection between regenerated endothelia and basal membrane, which more approximate the normal.8．Rats transplanted with CGRP-EPCs had a significantly higher survival than those given culture medium (control group) whereas delivery of EPCs only produced a survival in between which was not significantly different from shunt group.Conclusions1．EPCs could be cultured from human peripheral blood mononuclear cells with theinduction of proper cytokines and be further applied in the cell transplantation approach for their differentiation potential.2．The reconstructed expression plasmid containing the right sequence was successfully produced and purified and applied in ex vivo transfection of EPCs which secreted CGRP accordingly. 3．Intravenously administered EPCs will home in on damaged pulmonary vascularbeds and congregate there indicating the EPCs are capable of sensing the impaired tissues that need to be restructured. EPCs can also act as an effective therapeutic gene vehicle.4．Transplantation of CGRP-EPCs through intravenous injection can attenuate the elevated pulmonary hypertension in response to left-to-right shunt. Further more this method proves effective in restraining the hyperplasia of medial smooth muscle cells and reversing pulmonary vascular remodeling.5．These findings suggest that the therapy based on the combination of both CGRPgene and EPCs may be a potentially useful strategy for the treatment of pulmonary hypertensive disorders.