angiogenesis is a constant hallmark of multiple myeloma ( mm ) progression and has prognostic potential . we have previously isolated endothelial cells ( ec ) from bone marrow of patients with mm ( mmec ) . they show intrinsic angiogenic ability , because they rapidly form a capillary network in vitro , and extrinsic ability , because they generate numerous new vessels in vivo in the chick embryo chorioallantoic membrane ( cam ) . moreover , mmec secrete angiogenic cytokines , such as fibroblast growth factor-2 ( fgf-2 ) and vascular endothelial growth factor ( vegf ) , as well as matrix metalloproteinase-2 and 9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells . studies by rajkumar et al . , dominici et al . , and laroche et al . the bone marrow microenviromnent plays a crucial role in inducing the angiogenic response in mm [ 6 , 7 ] . reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells , namely haematopoietic stem cells , fibroblasts , ostoblasts / osteoclasts , ec , ec progenitor cell , t lymphocytes , macrophages and mast cells are mediated by an array of cytokines , receptors and adhesion molecules . these cells secrete angiogenic factors , such as interleukin-6 ( il-6 ) , vegf , fgf-2 , hepato - cyte growth factor / scatter factor ( hgf / sf ) , transforming growth factor- ( tgf- ) and tumour necrosis factor  ( tnf- ) [ 6 , 7 ] . here , we have attempted a fine characterization of the angiogenic response induced by mmec , as compared with monoclonal gammopathy of undetermined significance ec ( mgusec ) in the cam assay . we investigated the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implantedonthecamsurfacefrom8today12ofincu - bation and we studied the effects of mmec and mgusec on the expression of endogenous levels in the cam of vegf , fgf-2 , angiopoietin-1(ang-1 ) , hypoxia - inducible factor-1 ( hif-1 ) and endostatin by reverse transcriptase - polymerase chain reaction ( rt - pcr ) . mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described . fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell . the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions . the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively . the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) . blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x . means  1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data . total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining . the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) . level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin . pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) . preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) . intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) . six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] . after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide . the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst . peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film . control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load . stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) . mmec were obtained from 10 patients with mm and mgsuec were obtained from 10 patients with mgus , as previously described . fertilized white leghorn chicken eggs were incubated at 37c at constant humidity . on day 3 of incubation a square window was opened in the egg shell after removal of 23 ml of albumen so as to detach the developing cam from the shell . the window was sealed with a glass and the eggs were returned to the incubator . gelatin sponges ( gelfoam , upjohn company , kalamazoo , mi , usa ) were cut to a size of 1 mm3 and placed on top of a growing cam at day 8 incubation under sterile conditions . the sponges were then adsorbed with 2 l of cell suspension ( 18,000 cells per sponge ) of mmec or mgusec or with rpmi-1640 medium alone or supplemented with fgf-2 ( 200 g / ml ) ( r & d systems , abington , uk ) , used as negative and positive control , respectively . the angiogenic response was evaluated 96 hrs after the implants by means of a stereomicroscope connected to an image analyser system ( olympus italia , italy ) . blood vessels entering the sponges within the focal plane of the cam were counted by two observers in a double blind fashion at a magnification of 50x . means  1 standard deviation ( sd ) were evaluated for all the parameters and the statistical significance of the differences between the counts was determined by student 's t - test for unpaired data . total rna was extracted from 12 cam treated with ec obtained from six patients with mgus and six patients with mm and from six control cam using trizol reagent ( invitrogen , carlsbad , ca , usa ) according to the manufactures 's protocol . the concentration and the purity of the isolated rna were determined using a spettrofotometric analysis and the integrity of the rna was verified by means of electroforesis in a formaldehyde agarose gel followed by ethidium bromide staining . the cdna was then synthetized with 5 g of total rna using m - mlv and oligo ( dt)1820 as primer ( invitrogen , carlsbad , ca , usa ) . level of mrna expression of pro - angiogenic molecules vegf , fgf-2 , ang-1 , hif-1 and mrna expression of anti - angiogenic factor endostatin were evaluated by pcr using specific primers and the relative level of the each amplified transcripts were normalized to those of the housekeeping gene -actin . pcr reactions were performed in a final volume of 25 l containing 2 l of rt products , 10 picomoles of each primer and 1x of red - taq polymerase mix ( sigma - aldrich , milano , italy ) . preliminary experiments were performed to determine the appropriate conditions ( ta and specific number of cycles ) for semi - quantitative rt - pcr and transcript were amplified using different cycle numbers:25 cycles for -actin , 30 cycles for vegf , fgf-2 , ang-1 and for endostatin and 35 cycles for hif-1. pcr products were subjected to electrophoresis in a 1.5% agarose gel and ethidium bromide stained , and then photographed under an ultraviolet transilluminator ( amersham biosciences ) . intensity of bands was quantified as arbitrary optical density units ( od ) using the scion image software ( based on nih image ) . six cam treated with ec obtained from three patients with mgus and three patients with mm and three control cam were homogenized in ice cold buffer containing 250 mm sucrose , 10 mm tris - hcl , ph 7.4 and protease inhibitors [ 1g / ml leupeptin , 1g / ml pepstatin and 1 mm phenyl - methylsulfonyl fluoride ( pmsf ) ] . after homogenization in a potter apparatus and centrifugation at 1000 g for 10 min , the protein content of the supernatant was measured using a bradford assay ( bio - rad laboratories , hercules , ca , usa ) . for sds - page , samples ( 60 g / lane ) were solubilized in laemmli buffer , boiled at 90c for 10 min and runned on a 12% polyacrylamide . the proteins were blotted to a pvdf membrane ( millipore corporate , billeira , ma , usa ) and immunoblotting was performed using a monoclonal mouse primary antibody against endostatin ( novus biologicals , littleton , co , usa ) diluted 1:500 in tbst . peroxidase activity was revealed by chemiluminescence ( ecl kit , santa cruz biotechnology , santa cruz , ca , usa ) and blot was immediately exposed to x - ray film . control experiments were performed using a mouse - anti--actin ( abcam , cambridge , ca , usa ) to detect housekeeping protein -actin to normalize the protein load . stained bands were scanned and intensity was quantified using the scion image system ( based on nih image ) . the ability of mmec and mgusec to induce an angiogenic response in vivo was assessed with the cam - gelatin sponge assay . 1 ) . cam implanted with the medium containing fgf-2 or mmec gave significantly higher vessel counts and numerous allantoic vessels converging like spokes toward the sponges were recognizable ( table 1 and fig . when the sponges were loaded with the medium alone and with mgusec , physiologic angiogenesis was observed in the form of few allantoic vessels partly around and partly converging toward the sponge ( table 1 and fig . angiogenic response assessed by the chick embryo chorioallantoic membrane ( cam)-gelatin sponge assay p<0.001 versus rpmi-1640 and mgusec angiogenic activity in the chorioallantoic membrane ( cam ) assay . gelatin sponges loaded with fgf-2 ( a ) , mmec ( b ) , rpmi-1640 ( c ) and mgusec ( d ) were implanted on top of the cam on day 8 . macroscopic view of the cam on day 12 shows numerous allantoic vessels converging like spokes toward the sponge in a and b , while few allantoic vessels are recognizable in c and d. original magnification : a  d , x 50 . in order to evaluate the angiogenic effects of mmec and mgusec , mrna expression of pro - angiogenic factors including vegf , fgf2 , ang-1 , hif-1 and mrna expression of anti - angiogenic molecule endostatin the expression level of each transcript was compared to those detected in control cam . as shown in fig . 2 , treatment of cam with mmec and mgusec did not produce significant difference in the expression of pro - angiogenic factors mrna in comparison with control cam treated with medium alone . moreover , no statistical difference was found in vegf , fgf-2 , ang-1 , hif-1 and endostatin mrna content between mm and mgus treated cam ( p<0.05 ) . in contrast , the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam ( mgus od:120.113 and mm od:48.319.6 versus ctrl od:205.84  19.6 ; p<0.05 ) . expression levels of mrna coding for vegf , fgf-2 , ang-1 , hif1 and endostatin evaluated by semi - quantitative rt - pcr . transcript levels from the cam assay are referred to ec obtained from 6 mm and 6 mmgus patients and the error band represents the standard deviation of six experiments . western blot analyses were also performed to study whether the expression of endostatin changes in mmec and mgusec treated cam respect to control . as shown in fig . 3 , according to rt - pcr quantification , endostatin was significantly lower also at the protein level in both mgusec and mmec treated cam respect to control cam ( mgus od : 97.1410 and mm od : 52.313.6 versus ctrl od : 158.715.52 ; p<0.05 ) . immunoblotting analysis and quantification after western blot analysis of endostatin expression in cam treated with ec obtained from 3 mm and 3 mgus patients . mm plasma cells secrete vegf , whereas both vegfr-1 and vegfr-2 are markedly elevated in the bone marrow stromal cells , suggesting that a paracrine growth pathway mediated by vegf - activated stromal cells , secretion of il-6 on their part , and subsequent activation of plasma cells may occur . in active mm , we found secretion of the vegf - a isoform by plasma cells , overexpression of vegfr-2 by bone marrow microvessels and isolated ec and of vegfr-1 by the other stromal cells , secretion of vegf - c and vegf - d by stromal cells , and expression of their cognate receptor vegfr-3 by plasma cells , which suggests another paracrine loop of angiogenesis and tumour growth in mm . parallel studies by kumar et al . found secretion of vegf by several mm cell lines and fresh patients ' bone marrow plasma cells , as well as expression of vegfr-1 and vegfr-2 , which suggests multiple vegf - mediated autocrine pathways of tumour growth and paracrine stimulation of angiogenesis . when we studied the angiogenic potential of plasma cell suspension obtained from patients with active mm and implanted them onto the cam , we demonstrated a vasoproliferative response , significantly higher than that induced by cell suspension obtained from patients either with non - active mm or mgus . hgf / sf is yet another angiogenic factor identified in the culture medium of both human mm cell lines   and freshly isolated plasma cells . still other factors may be responsible for bone marrow angiogenesis in mm : the plasma cell secretion products , such as tgf- , il-1 , il-6 and il-8 ; granulocyte - colony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) and tnf- , secreted by bone marrow microenvironment cells recruited and activated by plasma cells . since human mm cell lines produce ang-1 that up - regulates the tie-2 cognate receptor in bone marrow ec , involvement of the ang-1/tie-2 loop in mm neovascularization has been demonstrated . conversely , mmec secrete angiogenic cytokines , such as fgf-2 , vegf and ang-1 as well as matrix metalloproteinase-2 and -9 ( mmp-2 and mmp-9 ) and express adhesion molecules facilitating dissemination of these cells . other studies , however , showed that the expression levels of vegf , fgf-2 , and their receptors were similar among plasma cells from mgus and mm , suggesting that increasing angiogenesis from mgus to mm is , at least in part , explained by increased tumour burden rather than increased expression of vegf / vegfr-2 by plasma cells . another possibility is that the inhibition of angio - genesis in mgus is lost with progression , hence that the switch from mgus to mm may involve a loss of an anti - angiogenic activity . our data are in accord with this statement , because the expression of endostatin mrna detected in both mgus and mm treated cam was significantly lower respect to control cam . the phenotypic switch to angiogenesis in tumour growth involves more than simple up - regulation of angiogenic activity and is thought to be the result of a net balance of positive and negative regulators , and our study suggests that angiogenic switch in mm , investigated by using an in vivo experimental model such as the chick cam , may involve loss of an endoge - nous angiogenesis inhibitor , namely endostatin .
abstractwe have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells ( mmec ) by using the chick embryo chorioallantoic membrane ( cam ) assay and by reverse transcriptase - polymerase chain reaction ( rt - pcr ) . 
 results showed that in the cam assay mmec induced an angiogenic response comparable to that of a well - known angiogenic cytokine , namely fibroblast growth factor-2 ( fgf-2 ) , while rt - pcr demonstrated that the expression of endostatin mrna detected in mm treated cam was significantly lower respect to control cam . 
 these data suggest that angiogenic switch in mm may involve loss of an endogenous angiogenesis inhibitor , such as endostatin .