| Objective:The aim of this thesis has been to investigate the relevance of tissue engineering strategies for the regeneration of the heart following MI. The aim of this thesis has been to investigate the relevance of tissue engineering strategies for the regeneration of the heart following MI. We have assessed the use of fibrin-based hydrogels to provide either VEGF or CD133-selected putative EPCs to the heart in order to stimulate neoangiogenesis. Specifically, we investigated 2 major questions: 1, Angiogenic effects of VEGF121 covalently bound to fibrin gel in non-infarcted and infarcted rat hearts, after 4 weeks of implantation. In particular we tested: 1) If VEGF121 covalently bound to fibrin gel could promote angiogenesis in the non-infarcted and infarcted rat hearts; 2) If this cell-demanded release of VEGF121 from fibrin gel could induce proper angiogenesis, without leading to hemangioma formation; 3) How far the angiogenic effect of VEGF121 can reach; 4) Which kind of vessel VEGF can promote; 5) Whether the angiogenic effect of VEGF121 can improve the heart function; 6) Whether the fibrin gel per se has some angiogenic influences to the rat hearts. 2, Possible vasculogenic or angiogenic effects of EPCs incorporated onto fibrin gels in the non-infarcted or infarcted rat hearts after 3 or 8 weeks of transplantation: 1) Whether EPCs delivered by fibrin could proliferate in the non-infarcted and infarcted rat hearts, incorporate to the neovessels and thus promote neovascularization; 2) Whether EPCs delivery to the heart can improve heart function.Methods: First of all that are Preparation of fibrin gel matrices, preparation of recombinantα2-PI1–8-VEGF121and Fibrin gel matrices formulated withα2-PI1–8- VEGF121. 1, A total of 28 male Sprague-Dawley (SD) rats(9 week-old, weight 300-350 g) were included in this experiment, 13 of them received left anterior descending artery (LAD) ligation to induce the left ventricular myocardial infarction (MI).15 non-infarcted and 7 infarcted and 6 non-infarcted rats were implanted with fibrin gels covalently conjugated with VEGF. As a control, 6 non-infarcted rats and 6 infarcted rats received empty fibrin gels; other 3 normal rats with no treatment at all were use as control for the histology studies. In the infarcted rats, implantations were performed immediately after the ligation. All rats were kept for 4 weeks and before sacrifice, the heart function was evaluated by echocardiography. Histological and immunohistochemi- cal study:Rats were sacrificed with intravenous injection of potassium chloride (2 ml KCl 15%) at the level of the femoral vein, in order to stop the heart in diastole. Hearts were rapidly excised, the cardiac cavities were rinsed with PBS to remove blood and thrombus, then the hearts were fixed with 10% formalin for 24 hours. Afterwards, the hearts were cut into 3 parts parallel to the atrioventricular groove before being dehydrated and embedded in paraffin. Blocks containing the sections in contact with the cardiac patch were cut into 3μm thickness slices and stained with hematoxylin and eosin. Consecutive sections were used for the immunohistochemical study. For the VEGF conditions, 3 in 60 sections were stained with anti-CD31 and 1 in 60 sections was stained with anti-α?smooth muscle actin (SMA) to detect the vessels. 3 in 60 sections per heart were stained with anti-CD31 and 9 or 12 photos were taken per section at a magnification of 200X. For the non-infarcted hearts, 2 zones picture by taken: under and border of the gel, i.e. near the gel and far from the gel, i.e. in the septum. And took six photos per zone. Similarly, for the infarcted hearts, 6 pictures were randomly taken in two newly-defined zones, i.e. in the ischemic border zone of the infarct (near the gel) and (in the septum). For vascular measurements, three parameters were determined: 1) total vessel density (total vessel number per mm2 of total tissue area), 2) ratio of total vessel number to myocardial area (total vessel number per mm2 of myocardial area) and 3) percentage of total vessel area (total vessel area per total tissue area, %). Total tissue area was defined as the total image area minus the interstitial space (white empty space), to exclude a bias due to differences in the occurrence of interstitial space (either natural or artifactual, for instance tissue laceration during processing). Then, myocardial area was defined as total tissue area minus total vessel area. To assess which kind of vessel VEGF could promote, vessels were directly assigned by the Metamorph Software to one of three groups:①capillaries (vessel diameter<10μm),②microvessels (10μm≤vessel diameter≤20μm) and③macrovessels (vessel diameter>20μm). 2, 27 male Sprague-Dawley (SD) rats (9 week-old, weight 300-350 g) were included in this experiment. The animals were divided into 3 main groups: group 1: non-infarcted rats (n=9); group 2: rats engrafted immediately after MI (n=9); and group 3: rats engrafted 1 week after MI (n=9). For each group, the rats were sacrificed either 3 weeks or 8 weeks after implantation. To prevent rejection of EPC-seeded gels, the rats were immunosuppressed by a cyclosporin A (CsA) delivered continuously via an osmotic minipump. For the evaluation of left ventricular function, transthoracic echocardiography was performed on the rats before sacrifice. Process of sacrificed and qictures taken of rat same with VEGF. The EPCs-gels transplanted hearts, 3 in 180 sections were stained with anti-CD34, every tenth of 180 sections were stained with anti-human CD34 and Tra-1-85 to detect the transplanted human cells. The EPCs study used the same sampling, i.e. 3 CD31- stained sections per heart and chose the simplified method: 1 zone per section was chosen (near the gel in the non-infarcted hearts and in the ischemic border zone of the infarct in the infarcted hearts) and 6 pictures per zone were taken. The method of vascular measurements is same with VEGF.Results: 1, At the first analyzed the effect of VEGF-engineered gels engrafted on rat left ventricles.To rule out the possibility that VEGF administrated at too high concentrations could increase the vascular permeability leading to leakiness and causing pericardial or pleural effusion, to examine the chest cavity surrounding the hearts upon their excision and did not see any sign of effusion neither in the chest nor in the pericardium. Some loose connective tissue was usually present between the heart and the chest, but no difference was found between the rats with VEGF gels and the rats with empty fibrin gels. Observed by H&E staining had a relatively normal morphology, and no hemangioma or vessel malformations were found. Observed slightly more loose connective tissue containing signs of an active neovascularization near the infarct area as compared to non-infarcted hearts, but this was not apparently different in rats receiving VEGF gels as compared to rats with empty gels.Then compared the total vessel density, the ratio of total vessel number to myocardial area, and the percentage of total vessel area in the three sampling areas (under the gel, at the border of the gel and far from the gel). In non-infarcted rats, no significant difference was found for these three parameters measured in the three sampling areas, and this was the case for either the VEGF-gel group or the empty-gel group.And then compared the same parameters between the VEGF-gel group and the empty-gel group respectively in the three sampling areas (under the gel, at the border of the gel and far from the gel). We observed a trend for an increased number of vessels in the VEGF-gel group but the difference was not statistically significant.However, when we combined the three sampling areas together, significant discrepancies in percentage of total vessel area (ANOVA F(2,59)=3.46; p<0.05) were observed between treated animals. Indeed, animals that received VEGF-fibrin gels had a significantly higher percentage of total vessel area than those with empty gels (p<0.05). The differences in percentage of total vessel area were closely correlated to differences in total vessel density: here the statistical significance was even higher (ANOVA F(2,59)=6; p<0.005) and animals exposed to VEGF also had a significantly higher ratio of total vessel number to myocardial area than those receiving empty gels (p<0.005). Since we did not see any significant difference among the three sampling areas (under the gel, at the border of the gel and far from the gel), we changed the sampling method to two zones (near the gel and in the septum), and took 6 instead of 3 images per zone. Given that the three parameters (vessel density, ratio of vessel number to myocardial area, and percentage of vessel area) gave very similar results, we used vessel density to perform all the vascular measurements. Since the total vessel density was not normally distributed, we were able to normalize it using the square root transform, namely getting a Gaussian distribution. As 36 observations were performed in each rat (3 sections, 2 zones, 6 pictures by zone), we took into account the clustering of the observation during the subsequent regression analysis and applied an ANOVA with a repeated measure design, both techniques taking into account the fact that the observations are not independent within the same rat.Considering all the groups together and using multiple regression, we predicted the square root of total vessel density from 3 independent variables: infarct (no or yes), localization of the sample (septum: no or yes) and treatment group (without treatment, empty-gel, VEGF-gel) and their respective interactions. The resulting model explained 25% of the variance in the square root of total vessel density, with none of the interaction terms being significant, and only one main effect remaining significant: the total vessel density in the VEGF-gel group being significantly higher than in both empty gel group (P=0.004) and without treatment group (P=0.010).We then compared the square root of total vessel density among the subgroups by ANOVA, taking into account that repeated observations were performed on the same rats. However, statistical significance was found in the differences among the VEGF-gel, empty gel and without treatment groups in the area near the gel for the non-infarcted rats when using multiple regression analysis (P=0.030). Considering all the 6 subgroups together for the non-infarcted hearts, square root of total vessel density analyzed by ANOVA shows a significant group effect (P=0.0057), but no zone effect and no interaction of zone with group. It was significantly higher in the VEGF-gel group than in the empty gel group (P=0.0206) or the group without treatments (P=0.0026). Again, the empty-gel group and the group without treatment were not different.To investigate which kind(s) of vessel(s) VEGF could promote, we classified vessels by Metamorph Software analysis into the following three groups: (1) capillaries (vessel diameter < 10μm), (2) microvessels (10μm≤vessel diameter≤20μm) and (3) macrovessels (vessel diameter>20μm). We calculated the following parameters: capillary density, microvessel density and macrovessel density. The capillary density in non-infarcted and infarcted rat hearts was significantly higher in the VEGF-gel group than in the empty gel group. While in non-infarcted hearts, no difference was observed between the areas near the gel and in the septum, regardless of the treatment, in infarcted rats, the capillary density in the VEGF-gel group was significantly higher near the gel as compared to the septum. Differences between non-infarcted and infarcted heart were not significant, neither in the VEGF-gel group nor in empty gel group. Then normalized the capillary density by calculating its square root (sqrt) (as described above). Using multiple regression (with rats clustering), we predicted the square root of capillary density from 3 independent variables: infarct (no or yes), localization of the sample (septum: no or yes) and treatment group (empty gel or VEGF-gel) and their respective interactions. The resulting model explained 24% of the variance in the square root of capillary density, with none of the interaction terms being significant and only one main effect being significant: sqrt of capillary density in the VEGF-gel group being significantly higher than in empty gel group (P=0.003).Compared the sqrt of capillary density among the subgroups by ANOVA, taking into account that repeated observations were performed on the same rats. Only the difference between the VEGF-gel group and empty gel group in the area near the gel was significant in infarcted rat hearts. Multiple regression analysis did not detect significant difference between the VEGF-gel group and the empty gel group in the area near the gel in the non-infarcted rat hearts.Again considering all the 6 subgroups together for the non-infarcted hearts, square root of capillary density analyzed by ANOVA was significant higher in the VEGF-gel group than in the empty gel group (P=0.0228,) but no zone effect and no interaction of zone with group were observed. For the macrovessel density, we used the same multiple regression model. The model explained 18% of the variance, with none of the interaction terms being significant except one main effect: macrovessel density in the VEGF-gel group was significantly higher than empty gel group (P=0.021). When the macrovessel density among the subgroups was analyzed by ANOVA, no significant difference was found between VEGF-gel group and empty gel group either in the non-infarcted rats (P=0.0586) or in the infarcted rats (P=0.2481). Measured macrovessels included both arteries and veins and we judged the sampling of 6 images per zone not sufficient to prevent a bias on the result interpretation, we scored the number of arteries in the whole left ventricle in 3 of 60 sections per heart shows no significant difference in the artery density between the VEGF-gel group and the empty gel group either in the non-infarcted or in the infarcted rat hearts. The echocardiography was performed before animal sacrifice and cardiac function was assessed according to the parameters measured on the M-mode tracing showed the difference of cardiac parameters such as LVDs, LVDd and FS between the VEGF-gel group and the empty gel group in non-infarcted and infarcted rat hearts. No significant difference was found between the VEGF-gel group and empty-gel group in the non-infarcted rats, but in the infarcted rats, a pronounced trend to have better heart function in the VEGF-group was detected even though it was not statistically significant. To get the significance, more rats are needed. 2, Transplantation of EPCs-seeded fibrin gels. Macroscopic views of 2 of 20 rat hearts implanted with EPC-seeded gels. Performed 180 sections in the proximity of the gel area and 1 every 10 were stained by H&E. Heart structure engrafted with either EPCs- gels or empty-gels were indistinguishable and relatively normal, with no visible hemangioma, vessel malformations or tumors.To detect the transplanted human EPCs or their progenies in the rat hearts and used anti-human CD34 antibody.CD34 marker worked nicely as positive control in human placenta sections. However, we were not able to retrieve CD34-positive cells in the rat hearts transplanted with EPCs-gels.It shows the immunohistochemical staining using anti-human CD34 antibody in non-infarcted as well as infarcted hearts. Given that no human CD34 (+) cells were ever found in any of the transplanted rats, we checked this antibody on EPCs-gels embedded for immunohistochemical examination. Surprisingly, EPCs were negative for anti-human CD34 antibody. Then chose another marker, the Tra-1-85 which is present on the surface of all human cells. Tested the anti-Tra-1-85 antibody on human placenta and on EPC-seeded fibrin gels It shown both placenta and EPCs in vitro were positively stained. Then used Tra-1-85 to detect the transplanted human EPCs or their progenies (EPC-derived ECs) in the EPCs-gel transplanted rat hearts. Unfortunately, no Tra-1-85-positive cells were detected in 1 every 10 sections examined over a total 180 sections. Given that no positive result was obtained by immunohistochemical examination, we tried preliminarily a technique of in situ hybridization (ISH) to identify human cells. ISH can detect Alu sequences in the human nuclei (stained in blue). In a first trial did not find any human cells in the 3-4 sections examined. Despite the lack of human EPC retrieval in the engrafted hearts, then decided to assess whether the transplanted EPCs had somehow an angiogenesis effect in the rat hearts. Total tissue area, total vessel and capillary number as well as their density were measured. No significant differences were found either between the rats with EPCs-gels and the rats with empty gels or among the different EPCs-gel groups.Evaluation of left ventricular function after EPC engraftment, there were not enough non-infarcted rats for the statistical analysis,so we compared only the infarcted rats engrafted with empty or EPC-seeded gels. Using ANOVA analysis, there was no significant difference among all the groups.Conclusions: 1, VEGF covalently immobilized in the fibrin gel could promote angiogenesis by increasing capillary formation in the non-infarcted and infarcted rat hearts, an effect more pronounced in the ischemic area of infarcted rat hearts. The study controlled sustained low dose release of VEGF prevented the formation of aberrant vessels. Infarcted rats with VEGF-gels have a trend to have better cardiac function compared to the rats with empty gels. Further studies with a higher number of rats are in process in order to show a significant impact of such strategy on the heart function. If the results presented in this work will be confirmed, fibrin gel matrices could constitute a future efficient vehicle for delivering angiogenic factors to the heart. 2, Although our EPCs results were not positive at this point, we will modify and improve our strategy of cell delivery through fibrin matrices and we are confident that such a cell delivery system will prove beneficial and effective in the future. |
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