CONCURRENT VASCULOGENESIS AND ANGIOGENESIS IN THE NORMAL HUMAN EMBRYO

ORIGINAL ARTICLES CONCURRENT VASCULOGENESIS AND ANGIOGENESIS IN THE NORMAL HUMAN EMBRYO Anc Cimpen 1, Johnnes Achench 2, Mrius Ric 1 ABSTRACT Four humn emryos were investigted (5, 7, 7 nd 8 weeks old). Specimens were completely emedded in prffin nd seril cross sections were performed on the entire emryos. Between 60 nd 186 slides were otined, ech contining three sections. Ech 10 th section ws stined with routine hemtoxylin-eosin method. The next step ws stiny for vimentin, CD34 nd smooth muscle cell ctin. Blood vessels were counted using the hot spot method t x200 mgnifiction. No lood vessels were found in the crtilge, liver, rin nd spinl cord. The lrgest numer of lood vessels were noticed in the lung, ventricle, nd kidney. The lrge mjority of these vessels were immture nd lot of single endothelil cells were noticed. Only rre mture lood vessels were demonstrted with smooth muscle cell ctin. CD34 ws highly specific, ut it did not stin ll endothelil cells. We concluded tht CD34 is less sensitive for endothelil cells of the emryo thn for endothelil cells found in mlignnt tumors (s we found in previous studies). It is estimted tht the phenotype of the endothelil cells during vsculogenesis nd ngiogenesis in emryo is different from tht descried in tumor ngiogenesis. Key Words: vsculogenesis, ngiogenesis, humn emryo, single endothelil cells, immture lood vessels, CD34 INTRODUCTION Vsculogenesis is the process of lood vessel formtion from lood islnds nd usully, it is found in its pure form only in the emryo. Vsculogenesis is multistep process tht involves the differentition of mesenchyml cells into ngiolsts nd then into endothelil cells. On the other hnd, some steps of this process re not completely understood. Few dt is ville on how long lood islnds persist, on the differentition of ngiolsts into endothelil cells, nd on the moment the lumen ecomes evident. Angiogenesis is the process of lood cell formtion from pre-existent lood vessels. 1-3 As the formtion of the lood vessel is lredy initited, the growth my continue s vsculogenesis or ngiogenesis is initited. Recently, Ashr et l., descried the ddition of circulting precursors of endothelil cells tht re eventully integrted into the wll. 4 This mechnism is likely to e found only in dult ngiogenesis fter 1 Deprtment of Histology Victor Bes University of Medicine nd Phrmcy, Timisor, Romni 2 Medizinische Hochschule of Hnnover, Germny Correspondence to: Anc Cimpen Victor Bes University of Medicine nd Phrmcy, Timisor, Romni P-t E. Murgu nr. 2, Timisor the complete differentition of the one mrrow. Tumor ngiogenesis is nowdys trget for therpy. The network of lood vessels found in mlignnt tumors does not initite the tumor ut it is essentil for its growth. As demonstrted mny yers go y Folkmn et l., tumors without lood supply do not grow more tht 3 mm in their lrger dimeter. 5,6,7 Tumor lood vessels were clssified s single endothelil cells, immture (without pericytes or smooth muscle cells) nd mture (with pericytes or smooth muscle cells). 8 The lrge mjority of studies tht focused on vsculogenesis nd ngiogenesis were performed on niml models. Our purpose ws to identify different steps of vsculogenesis nd ngiogenesis in the norml humn emryo nd to check for similr nd different spects compred with tumor ngiogenesis. We focused on the numer of smll lood vessels nd on their type, ccording to the clssifiction pulished y Gee et l. 8 We lso investigted the cell popultion round the newly formed lood vessels tht helps to understnd the ddition of pericytes nd/or smooth muscle cells to the lood vessel wll. MATERIAL AND METHODS We investigted four humn emryos (5, 7, 7, nd 8 weeks of ge) tht were entirely sliced (step sections) Anc Cimpen, et l. 219

t 5 micrometer thick. Seventy-five sections from ech cse were stined with the routine hemtoxylin-eosin method for the study of lood vessel morphology. Additionl sections from ech cse were immunostined for vimentin (clone V9) (25 slides), smooth muscle cell nti ctin (clone 1A4) (25 slides), nd CD 34 (clone BI3C5) (25 slides). Antigen retrievl ws performed in ll cses using microwves nd the uffer citrte ph 6, for 20 to 30 minutes. The working system ws LSAB2 for vimentin nd smooth muscle cell ctin nd ChemMte for CD34. The finl rection product ws visulized with DAB; therefore, ny positive rection ws stined in rown color. Nuclei were counterstined with modified Lillie s Hemtoxylin. All regents were from DAKO Corportion Denmrk. Blood vessels were counted using the Hot Spot method t x200 mgnifiction tht reflects field of 0.63 cm 2 (choosing the field of highest density). Blood vessels were then inspected for positive rection with smooth muscle ctin nd CD34. Blood vessels with ll spces lined y CD34 positive cells were took into ccount. Isolted CD34- positive cells were considered single endothelil cells. The numer of lood vessels/x200 field ws grded s follows: 0 sent, + less thn 3, ++ 3 to 10, nd +++ more thn 10. RESULTS On hemtoxylin eosin stined preprtions the presence nd numer of lood vessels lrgely depends on the ge of the emryo. In the 5-week old emryo the primordi of the hert nd of lrge lood vessels were lredy developed. Very rre smll lood vessels nd lood islnds were found nd occsionlly, nucleted red lood cells were noticed within the mesenchyme. In the 7-week old emryo (27 mm long), mny lood vessels were found minly in the mesenchyml tissues. The highest density ws noticed in the mesenchyme of the primitive lung, hert nd in the undifferentited tissue locted round crtilges (Fig. 1). Few lood vessels were found in orgns like liver, trche or the primitive intestine. Smll lood islnds were found in the mesenchyme s ggregtes of ngiolsts nd nucleted red lood cells (Fig. 1). Only rre cells tht could e ngiolsts were found t the periphery of these islnds (Fig. 1c). Angiolsts or lessdifferentited endothelil cells sprouting from these islnds were oserved in lmost ll cses (Fig. 1d). It ws very difficult to identify single endothelil cells on slides stined with hemtoxylin-eosin. Moreover, in mny cses it ws difficult to discriminte etween mture nd immture lood vessels. The pprent distriution nd numer of single endothelil cells, immture nd mture lood vessels re shown in Tle 1. c d Figure1. The endothelium of the ventricle continues with the endothelium of lood vessels tht penetrte deep into the primitive myocrdium (). Blood islnds with ggregtes of committed mesenchyml cells nd nucleted red lood cells (). Angiolsts (c). Sprouting of ngiolsts from the wll of smll lood vessel (d) 220 220 TMJ 2003, Vol. 53, No. 3-4

CD34 ws chosen to identify endothelil cells s it is highly sensitive, in spite of the fct tht it is less specific thn CD31. The internl positive control ws the endothelium lredy developed within the ort. In the 5-week old emryo, endothelil cells of the ort were lmost cuoidl nd more thn 90% showed strong positive rection of the cytoplsm (Fig. 2). In the 7- nd 8-week old emryos ll flt endothelil cells were strongly positive (Fig. 2). The nervous tissue of the rin, spinl cord nd peripherl nerves were negtive. We lso found negtive rection in the liver, drenl nd crtilge. The finl rection product ws intensely stined in rown with diffuse cytoplsmic pttern. All endothelil cells nd their precursors expressed CD34, except those of smll lood vessels of the outer lyer of the primitive intestine (Fig. 3). Only rre isolted cells, spindle or stellte in shpe, of the mesenchyml tissue were slightly positive with grnulr cytoplsmic pttern (Fig. 3). Therefore, the CD34 immunorection ws considered specific enough to numer lood vessels nd to evlute their type. Figure 3. Primitive intestine with positive lood vessels in the middle of the wll nd negtive t the periphery (). Isolted spindle cells with wek grnulr expression of CD34 () Figure 2. CD34 expression in the endothelium of the ort. 5-week old emryo (). 7-week old emryo () Few smll lood vessels were found in nerve gngli, the outer lyer of the trche, the midlyer of the esophgus nd the medull of the thymus. Mny lood vessels were found in the lung (Fig. 4), kidney (Fig. 4), hert (Fig. 4c), smll intestine (Fig. 4d), nd thyroid. There were positive lood vessels with evident lumen, endothelil cells sprouting from the wll s well s single endothelil cells. Blood islnds positive for CD34 were found only in the mesenchyml tissue of the 5-week old emryo (Fig. 5). Specific cells were rrnged in smll nests or cords; ngiolsts, nucleted red lood cells nd their precursors were negtive for CD34. Only rre remnnts of these islnds were noticed in 7- nd 8-week old emryos. A rich network of smll lood vessels ws noticed in the mesenchyml tissue (Fig. 6). The lrgest numer of lood vessels ws found in the mesenchyme of the hed. Between vessels with evident lumen mny single endothelil cells were found (Fig. 7). Sprouting of endothelil cells ws oserved from remnnt islnds nd from preexisting vessels s well (Fig. 8, ). Anc Cimpen, et l. 221

Figure 5. Blood islnd with strong positive rection in the mesenchyme of the 5-week old emryo c Figure 6. The rich network of lood vessels of the mesenchyml tissue d Figure 7. CD34 positive isolted cells Figure 4. CD34 expression in the lung (), kidney (), hert (c) nd smll intestine (d) Committed mesenchyml cells were concentrted minly round the crtilge model of future ones. Numerous smll nd intensely rnched lood vessels were found close to the extrcellulr mtrix of the hyline crtilge t this level. The rection for smooth muscle cell ctin in the 5- week old emryo ws negtive except for very wek positive stining in the medi of lrge xil lood vessels. 222 222 TMJ 2003, Vol. 53, No. 3-4

All smll lood vessels, single endothelil cells nd lood islnds identified with nti-cd34 were consistently negtive. Therefore, it ws concluded tht ll smll lood vessels found t this stge of development were of immture type nd the differentition of smooth muscle cells ws only t the eginning. mjority of CD34 positive elements were single endothelil cells (SEC). The highest numer of lood vessels with endothelium positive for CD34 ws found in the lung, kidney nd ventricle. Blood vessels positive for ctin were no more thn 3 per x200 microscopic field (mesenchyme nd lung). This mens tht the lrge mjority of lood vessels were of immture type. Aprt from results otined in the periderm, ventricle nd drenl, the numer of single endothelil cells previled over the numer of lood vessels with evident lumen. Figure 8. Sprouting of endothelil cells from remnnt lood islnd (), nd from n lredy developed vessel () In the 7- nd 8-week old emryos the strongest rection ws found in the outer muscle lyer of the esophgus; with moderte intensity in the medi of lrge lood vessels. Blood vessels were very rrely stined in the mesenchyml tissue s well s in ll other orgns. Moreover, in mny cses the lyer of positive cells ws discontinuous even in vessels with reltively lrge lumen (Fig. 9). Compring these results with those otined with CD34, we concluded tht the lrge mjority of lood vessels in the humn emryo re of n immture type. Mture lood vessels with smooth muscle cells were rrely well represented in the medi. The type of the lood vessels ws demonstrted y compring results otined with nti-cd34 with those otined with ntismooth muscle cell ctin. As it is shown in Tle 1, in mesenchyml tissue, developing one, the wll of the trche nd esophgus the lrge Figure 9. Blood vessel with smooth muscle cell lyer (), nd with discontinuous muscle/pericyte investment () Tle 1.The distriution nd numer of single endothelil cells nd lood vessels Tissue SEC / CD34 Lumen/CD34 Totl/CD34 Actin Periderm 6 (18.1%) 27 (81.8%) 33 1 Mesenchyme 25 (62.5%) 15 (37.5%) 40 3 Muscle 9 (56.2%) 7 (43.8%) 16 1 Bone 7 (70.0%) 3 (30.0%) 10 0 Esophgus 23 (85.1%) 4 (14.9%) 27 0 Thyroid 24 (68.5%) 11 (31.5%) 35 0 Trche 8 (80.0%) 2 (20.0%) 10 0 Ventricle 15 (21.4%) 55 (88.6%) 70 2 Lung 60 (50.0%) 60 (50.0%) 120 3 Kidney 30 (37.5%) 50 (62.5%) 80 2 Adrenl 0(0%) 10 (100%) 10 0 Intestine 16 (40.0%) 24 (60.0%) 40 2 Nerve gngli 7 (50.0%) 7 (50.0%) 14 0 Anc Cimpen, et l. 223

The rection for vimentin ws found positive in mny mesenchyml cells, especilly in the re of hed nd neck. Vimentin ws not found in single endothelil cells nd wek rection ws noticed in the wll of some lood vessels. It is not good mrker for estimting vsculogenesis nd ngiogenesis, ut informs out the good qulity of fixtion nd emedding tht llows further interprettion. DISCUSSIONS Vsculogenesis is the process of lood vessel formtion from the mesenchyme. Usully, this process is found only in the emryo, nd the formtion of the vsculr network is completed y ngiogenesis. In the emryo, vessels formed from splnchnopleuric mesoderm cells re clled ngiolsts. They fltten nd join together to form ngiocysts, which in turn link together to form tuulr ngiolstic cords. The ngiolstic cords form ngiolstic plexuses tht continue vsculogenesis, egin ngiogenesis or interclte mesoderml cells into the wlls of existing vessels. 9 In oth vsculogenesis nd ngiogenesis during the mturtion of the vessels wll, there re involved endothelil cells, murl cells nd the extrcellulr mtrix. This process implies prolifertion, ddition, rnching nd speciliztion on locl demnds. 10 Filure of mturtion of lood vessels is often ssocited with vrious pthologicl conditions. Angiogenesis is the process of lood vessels formtion from preexistent lood vessels nd it ws reported in norml tissue, wound heling, nd lso in mlignnt tumors. It is multistep process tht overlps in prt with vsculogenesis. Concurrent vsculogenesis nd ngiogenesis re noticed in the emryo, ut t present time there re few reports regrding their ehvior in the humn emryo. During emryonic development, the vsculr network is formed y vsculogenesis nd the most powerful stimulus is the vsculr endothelil growth fctor (VEGF). VEGF not only stimultes the prolifertion of endothelil cells ut lso their mturtion. CD31+, CD34+ nd VEGF receptor-2 positive ngiolsts form vsculr plexus tht gives rise to the dorsl ort, crdinl vein nd to the vessels of the yolk sc. 11,12,13 This dt is confirmed y our findings ecuse these lrge vessels were lredy found in the 5-week old emryo nd they were well developed in the 7- nd 8- week old emryos. Moreover, no other lood vessels were noticed in the 5-week old emryo; however, lot of CD34+ lood islnds were identified. Mturtion of lood vessels mens stiliztion of the structure of the wll y ddition of murl cells (pericytes or smooth muscle cells). 14 As Kerel et l. pointed out, the difference etween mture nd immture lood vessels resides in the presence of pericytes or smooth muscle cells tht stilize the wll. Immunohistochemistry for n endothelil mrker comined with nti-smooth muscle cell ctin cn, therefore, discriminte etween mture nd immture vessels. 15 We found tht in the humn emryo over 90% of cells stined for CD34 elonged to SECs nd immture vessels, nd only few hd concomitnt positive rection for ctin. Moreover, in mny orgns (esophgus, trche, thyroid, drenl) mture lood vessels were not found. In others, like the liver, neither mture nor immture lood vessels were found. Our results re similr to dt pulished y other uthors tht investigted vsculogenesis in the humn emryo. 16 In mny mlignnt tumors, ngiogenesis reproduces lmost ll steps of the sme process found in the emryo. The difference consists minly in the numer of endothelil sprouts tht re more numerous in the emryo s we found in the lung, ventricle, esophgus nd kidney. In tumor ngiogenesis the presence of SECs, immture nd mture lood vessels is well demonstrted. In oth emryo nd mlignnt tumor the vsculr network is necessry for further growth. The question is: do endothelil cells hve the sme phenotype in oth conditions? To nswer this question it is importnt to discuss the sensitivity nd specificity of endothelil mrkers. Von Willernd fctor is highly specific ut less sensitive ecuse it cn e demonstrted only in the cytoplsm of differentited endothelil cells tht contins Weiel-Plde odies. 17 This is why we did not introduce this method in our study. The lrge mjority of studies on this topic showed tht CD34 is the most sensitive mrker of the endothelil cell, in spite of the fct tht it is less specific. Anti-CD34 ntiody clone QBEnd10 rects with endothelil cells nd their progenitors ut it lso lels hemtopoietic progenitors nd dendritic cells. Dendritic cells re sent in the humn emryo nd mture lood cells do not express CD34. Therefore, we considered CD34 specific enough to identify nd count lood vessels. On the other hnd, we found negtive rection for CD34 in lood vessels of the outer lyer of the primitive intestine, in spite of the fct tht we clerly noticed lood vessels with lumen lined y endothelium. This oservtion rises the prolem of CD34 sensitivity tht seems to e lower for the humn emryo thn previously thought. We reported high sensitivity for CD34 in tumor ngiogenesis (prostte cncer, urothelil crcinom, nd rest cncer). As expected, n intensely 224 224 TMJ 2003, Vol. 53, No. 3-4

positive rection (endothelium nd sprouts) ws noticed in ll conditions mentioned ove. Therefore, we cn conclude tht differences etween endothelil cells of the emryo nd those found in tumor ngiogenesis exist even t the level of the CD34 expression. For tht reson, we elieve tht the evlution of vsculogenesis nd ngiogenesis implies the comprison of CD34 nd CD31 expression. CONCLUSIONS CD34 is highly sensitive nd specific enough to identify lood vessels ut it ws not expressed y ll endothelil cells of the humn emryo Fetures of vsculogenesis do not completely overlp ngiogenesis In the 5-week old emryo vsculogenesis predomintes; in 7-week old emryo predomintes ngiogenesis Single endothelil cells nd immture lood vessels were the most numerous; only rrely lood vessels stined for smooth muscle cells ctin Immunohistochemistry for CD34 nd smooth muscle cell ctin discrimintes etween mture nd immture lood vessels Only some steps of the vsculo- nd ngiogenesis found in the humn emryo re similr with those found in tumor ngiogenesis REFERENCES 1. Weidner N, Folkmn J, Pozz F, et l. Tumor ngiogenesis: new significnt nd independent prognostic indictor in erly-stge rest crcinom. J Ntl Cncer Inst 1992;84:1875-87. 2. Ymzki PE, Ae S, Tgekw H, et l. Tumor ngiogenesis in humn lung denocrcinom. Cncer 1994;74:2245-50. 3. Zetter BR. Angiogenesis nd tumor metstsis. Ann Rev Med 1998;49:407-24 4. Ashr T, Msud H, Tkhshi T. Bone mrrow origin of endothelil cell progenitor cells responsile for postntl vsculogenesis in physiologicl nd pthologicl neovsculriztion. Circultion Res 1999;85:221-8. 5. Folkmn J. Tumor ngiogenesis: therpeutic implictions. N Engl J Med 1971;285:1182-6. 6. Folkmn J. Anti-ngiogenesis: new concept for therpy of solid tumors. Ann Surg 1972;175:409-16. 7. Folkmn J. The vsculriztion of tumors. Sci Am 1976;234:58-73. 8. Gee MG, Procopio WN, Mkonnen S, et l. Tumor vessel development nd mturtion impose limits on the effectiveness of nti-vsculr therpy. Am J Pthol 2003;162:183-93. 9. Clever O, Melton DA. Endothelil signling during development. Nture 2003; 9(6):661-8. 10. Folkmn J. Angiogenesis reserch: from lortory to clinic. Forum 1999; 9:59-62. 11. Crmeliet P. Mechnisms of ngiogenesis nd rteriogenesis. Nt Med 2000;285:389-95. 12. Jin RK. Moleculr regultion of vessel mturtion. Nture 2003;9(6):685-93. 13. Jin RK, Sfkhsh N, Sckell A, et l. Endothelil cell deth, ngiogenesis, nd microvsculr function fter cstrtion in n ndrogen-dependent tumor: role of vsculr endothelil growth fctor. PNAS 1998;95:10820-5. 14. Nguyen LL, D Amore PA. Cellulr interctions in vsculr growth nd differentition. Int Rev Cytol 2001;204:1-48. 15. Kerel RS. Tumor ngiogenesis: pst, present nd the ner future. Crcinogenesis 2000;21:505-15. 16. Isner MJ, Ashr T. Therpeutic ngiogenesis. Front Biosci 1998;3:49-69. 17. Shih SC, Roinson GS, Perruzzi CA, et l. Moleculr profiling of ngiogenesis mrkers. Amer J Pthol 2002;161:35-41. Anc Cimpen, et l. 225