Study On Target Therapy Of Leukemia By Anti-VEGF

It is an important field in the biological targeted therapy to therapy tumorwith the method of antiangiogenesis. Significant progressions have been achievedin the studies on solid tumor therapy by blocking vascularization. Someangiogenic inhibitors have been on the market, which gained anticipative effectson the solid tumor therapy. These good effects intrigued people's interesting onexploring leukemic therapy by using antiangiogenesis. Leukemia is ahematopoietic malignancy, deriving from bone marrow, which is different fromsolid tumor. Can antiangiogenesis get expected curative effects is the mainlycontrary?Many recent findings about this problem showed that there is also apivotal role for angiogenesis in the pathophysiology of leukemia. Bone marrowmicrovessel density in patients with leukemia was markedly increasing, eitherleukemic cell lines or primary leukemic cell has an excessive expression of bothVEGF and VEGFR, resulting in the generation of both autocrine and paracrineloop that not only support hematological malignancies cell proliferation but alsostimulate endothelial cell proliferation, which make bone marrow microvesseldensity (MVD) increase. During the proliferation of endothelial cells, in their turn,endothelial cells activated by VEGF trigger activity with subsequent release ofother growth factors, such as GM-CSF,IL-6,IL8 and many other cytokines. Therelease of these growth factors by the endothelial cells leads to induction ofleukemic cell proliferation through the paracrine loop. The proliferation ofleukemic cell in response to VEGF is triggered by intracellular signaling afterVEGF binding to the receptors on their own surface or binding to the receptors ofendothelial cells. Base on the angiogenesis theory, blocking any link of the signaltransduction initiated by VEGF and VEGFR in order to inhibit the biologicresponses of VEGF will provide more insight to help us to cure leukemia. By now,in targeting VEGF, inhibition of leukemia has entered the stage of clinical trials.To establish the objective estimated system of leukemic therapy throughanti-VEGF, further to evaluate the clinical significance, in our study, we tested thelevel of VEGF in both plasma (B-VEGF) and bone marrow (BM-VEGF), andbone marrow microvessel density (MVD). Specimens were collected from thefollowing four kind of typical patients, i.e. innocence anemia (iron deficiencyanemia, IDA,megaloblastic anemia, MA),newly diagnosed acute myelocyticleukemia (AML-NT),non-Hodgkin's lymphoma (NHL),free disease survivalacute myelocytic leukemia (AML-FDS), all the data from the four groups wascompared with each other simultaneously to reveal the clinical significance.Meanwhile, we innovated on the method of checking BM-MVD, in our studybone marrow MVD in plastic-embedded section was examined usingacetone-fixed bone marrow tissues embedded in glycol-methacrylate (GMA) resin.This technique labeled the microvessels in bone marrow with vWF as the antigenof vascular endothelial cells, not only the number of the bone marrowmicrovessels could be counted, but also the morphologic difference of endothelialcell could be observed distinctly among single endothelial cell, endothelial cellcluster, and endothelial cell of forming vessel. BM-MVD in innocent andmalignant hematopathy were different from one another on both quantity andmorphology. The results what we get have the characteristics of reliability,stability and well reproducibility, also the operation of the method is simple,which is convenient for the clinical application. This is a innovative technique, wehave not find the same report yet either internal or overseas.The consequence demonstrated that 1,a significant increase of B-VEGF innewly diagnosed AML,NHL patients but not in AML-FDS as compared with thecontrols, indicating B-VEGF can reflecting the pathological status of the body, thedetecting of B-VEGF has some clinical sense. Especially, B-VEGF level ofAML-DFS patients were also higher than normal controls, that remaindered us tothink highly of anti-VEGF therapy after long term remission of leukemia. As soonas the angiogenesis was inhibited, the tumor should be getting into quiescence.Nevertheless B-VEGF level in newly diagnosed AML patients was noticeablelower than what in the innocent anemia groups. This result maybe because of thatthe serum VEGF level is influenced by all kinds of factors, B-VEGF was notsuitable as the evidence absolutely for the estimation of leukemia. The result alsosuggested that B-VEGF was increasing in AML-FDS excluding any explainablereasons will signify relapse of the disease;2,There is remarkable increase inBM-VEGF and BM-MVD in newly diagnosed AML as compared with the otherthree groups, then BM-VEGF and BM-MVD could better reflect the invasion ofleukemia, which may be more important in contrast with B-VEGF;3,B-VEGF,BM-VEGF , BM-MVD were all increased in innocent anemia patients,consequently anemia was a unassailable influencing factor in the hemopoieticmicroenvironment, hypoxia led by durative anemia state induces secretion ofVEGF in normal tissue cell further initiates the angiogenesis. As a result, it isimportant to correct anemia,control infection during the course of therapy ,allabove measure is more significant for the disease recovery. We next to identify thedifference among B-VEGF,BM-VEGF,BM-MVD in AML patients, thenconclude that what BM-MVD was remarkable decreased after complete remissionwas the most obvious change, especially in AML-FDS group, so we can considerBM-MVD as a curative judgement for leukemia. As shown in our study, wecompared B-VEGF,BM-VEGF,BM-MVD with each other, above all regardBM-MVD as a curative judgement for leukemia, to reveal the clinical significanceof each data in various hematopathy or at various phase of one disease. Thisestimation system is a director for targeting anti-VEGF, we have not find anyreport so far.The convincing evidence that the VEGF concentration in bone marrow ishigher than that in plasma, and there is a direct relationship between BM-MVDand the prognosis of disease, demonstrated that the hemopoietic microenvi-ronment play a pivotal role in the leukemia therapy, that will give us an idea toapply directly the anti-VEGF agent to bone marrow can obtain better effects. Wecan get high concentration of sFlt-1 in bone marrow by means of transfectingsFlt-1 gene to bone marrow stroma cells. The sFlt-1 secreted by transfected cellsis capable of neutralizing the angiogenesis activity initiated by VEGF. sFlt-1 as ahigh selective neutralizing antibodies provide an ideal treatment for leukemia. Thefirst step we must identify whether sFlt-1 can inhibit the proliferation of leukemiccells without influencing the normal hematopoietic cells before transfectionexperiment. In the study ,we choose Reverse transcription-polymerase chainreaction (RT-PCR) ,Flow cytometer and enzyme-linked immunosorbent assay(ELISA) to identify the expression of VEGF and VEGFR-1 in K562,HL60,U937and bone marrow long-term culture-initiating cells (LTC-IC), the outcome showedthat K562,HL60 and U937 cell lines expressed VEGF as well as its receptor,K562 line owned the highest expression rate either in VEGF or VEGFR-1,expression rate of HL60 line was the second, U937 line had a lower level ofVEGF and VEGFR-1, nevertheless LTC-IC scarcely expressed VEGF andVEGFR-1. sFlt-1 was added to the culture system after plating the same amountof these leukemic cell lines, the consequence demonstrated that the inhibition onK562 and HL60 cell lines was as bigger as increasing the dose of sFlt-1, there wasa significant difference on inhibition ratio when adding various concentration ofsFlt-1, p<0.05. These data indicated the inhibition of sFlt-1 on leukemic cell lineswas concentration dependent. Instantaneouskly, the ration of tumor inhibition on24 hour, 48 hour and 72 hour time point was detected respecitively, the resultshowed the tumor suppressing effect was most intensively on 48 hour point., thatindicated the inhibition of sFlt-1 on leukemic cell lines was also time dependent.That means continue administer of sFlt-1 was necessarily for anti-VEGF therapy,it maybe the reason why the study on antiangiogenesis therapy of sFlt-1 are all bythe means of transgene methods. As control group, the proliferation of LTC-ICwas scarcely influenced. Based on the above findings, proliferation of normalbone marrow cells can not be affected when the activity of VEGF is suppressed,so, it is feasibility to transfect sFlt-1cDNA to bone marrow stroma cells forleukemia therapy.The objective of our study is to transfect the cDNA of sFlt-1D4 to bonemarrow stroma cells and gain the stable protein level of sFlt-1, we construct itsrecombinant eukaryotic plasmid pcDNA3-sFlt-1D4, then transfected pcDNA3-sFlt-1D4 into bone marrow stromal cells by Lipofectamine 2000, and the bonemarrow stromal cells expressing sFlt-1D4 can be secreted into cell culturesupernatants, which was prepared for the further experiment on murine. Theprevious researches showed that VEGF-binding domain with FLT-1 exist on the1-3 (Ig)-like domain of FLT-1 in the extracellular domain, VEGF binding to sFlt-1D3 domain is similar with VEGF binding to full-length sFlt-1.It is known that 2ndIg-like domain in Flt-1 carry a high affinity binding site for VEGF, 1st and 3rdIg-like domains in Flt-1 enhance the binding of Flt-1 to VEGF , 4th Ig-likedomain relate to the forming of dimer, which is essential for the signaltransduction .According to the above concept, we choose 1-4 Ig-like domains astargeted gene to amplify, not only exerting the high-affinity of transfectantproduction to VEGF, but also reducing the unnecessarily wastes. The result of theRT-PCR demonstrated that there was strap with 1400bp in length in bone marrowstroma cells transfected with pcDNA3-sFlt-1 D4 but was no target straps inempty-plasmid pcDNA3 cells and stroma cells without being transfected targetgene. We next to examine the sFlt-1 protein concentration in cells culturesupernatants after transfection at various time by ELISA, We found that thesFlt-1D4 protein could be detected by 24 hour after transfection, the rush hour ofsecretion was at the point of 48 hours, the high level protein was also detectableby 4 weeks, so we can get the stable protein production secreted by bone marrowstroma cells transfected with sFlt-1D4cDNA. Inhibition of sFlt-1D4 secreted bytransfectant cells on the proliferation of K562 was assessed via MTT assay andELISA. Transfectant cells culture supernatants by 24hours,48hours,1week,2weeks,3weeks and 4weeks were added to the K562 culture system, then theproliferations of K562 were suppressed to a certain extent in each group, therewas a significant difference compared with control groups. The result of MTTassay was consistent with that of ELISA method. All these results indicated thatbone marrow stromal cells which transfected with recombinant plasmid pcDNA3-sFlt-1D4 could secrete sFlt-1D4 and inhibite the function of VEGF. It is thefoundation for the further experiment on murine for leukemia therapy.