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First published online 8 March 2006
doi: 10.1242/dev.02315

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Vascular wall resident progenitor cells: a source for postnatal vasculogenesis

Elvin Zengin^1,*, Fariba Chalajour^2,*, Ursula M. Gehling³, Wulf D. Ito^4,5, Hendrik Treede², Heidrun Lauke¹, Joachim Weil⁵, Hermann Reichenspurner², Nerbil Kilic³ and Süleyman Ergün^1,

¹ Center of Experimental Medicine, Institute of Anatomy I, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
² Department of Cardiac Surgery, University Hospital Hamburg-Eppendorf, Hamburg, Germany,
³ Department of Hematology and Oncology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
⁴ Department of Cardiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.
⁵ Department of Internal Medicine, University of Schleswig-Holstein, Campus Lübeck, Germany.

View larger version (87K): [in a new window]   Fig. 1. Mural CD34+ cells in HITA wall. Besides the expected positive staining for CD34 in the endothelial layer (EC), CD34+ cells were found at the transition area from the smooth muscle cell (SMC) to adventitial layer (ADV) (A). Higher magnification reveals the close proximity of these cells to the external elastic membrane (arrows) of the vascular wall (B). FACS analyses on cells isolated from HITA wall after removal of mature endothelial cells positive for CD105 confirm the existence of mural CD34+ cells that are partially positive for von Willebrand Factor (vWF) (C,D). (E,F) Immunostaining for -smooth muscle actin revealed a clear staining of smooth muscle cells in the HITA wall (SMC) (E) and in the wall of vasa vasorum (arrows in F) (F) of HITA. No staining is visible in the CD34+ zone of HITA wall, shown at higher magnification in F.

View larger version (98K): [in a new window]   Fig. 2. Capillary sprouts from the HITA wall. HITA rings embedded in collagen gel or in matrigel show extensive capillary sprouting into the lumen (A) and outside the rings (B). Light microscopic studies on paraffin wax sections and electron microscopic studies on fine sections of HITA after the ring assay demonstrate the formation of tubular structures (arrowheads) in the lumen and outside the rings (C). The formation of capillaries (Cap) is also confirmed by electron microscopic analysis (D). New capillaries develop in close contact with mature endothelial cells based on the internal elastic membrane (arrow).

View larger version (95K): [in a new window]   Fig. 3. Capillary formation by and in vitro maturation of CD34+ VW-EPC. Immunostaining on sections of paraffin wax-embedded HITA rings after being subjected to ring assay demonstrates that intraluminar new capillaries exhibit both CD34 (A) and CD31 (B), whereas capillary outgrowth within the wall of and outside the HITA rings stains only positive for CD34 (arrows) (C). CD31 is detectable in vasa vasorum (arrows) (D) but not in new vessels at this stage of vascular formation. Capillarization through the entire vascular wall, as indicated by positive staining for CD34, occurs when rings were cultured for more than 14 days (E). Cells located in the CD34+ vasculogenic zone (arrows) and cells forming new tubes within the HITA lumen (arrowheads) exhibit immunostaining for VEGFR2 (F) and TIE2, the receptor for Ang1 and Ang2 (G). The endothelial marker VE-cadherin is present in mature endothelial cells lining the vascular lumen and in the newly formed vessels in the lumen (arrowheads) (H). Remarkably, VE-cadherin is also visible at the cell-cell contacts (arrowheads) of new vessel sprouts outside the rings (I). Immunostaining for -smooth muscle actin after the ring assay revealed only few positive cells within the adventitial layer (arrowhead) (J) and also within the collagen gel outside the rings (arrowheads) (K) that mostly tightly associated to the endothelial tubes (indicated by broken lines) or to single cells (arrow). (C,D,E,H,I,J,K) Counterstaining with Calcium Red; (A,B,F,G) counterstaining with Hematoxylin.

View larger version (69K): [in a new window]   Fig. 4. CEACAM1 expression in new endothelial sprouts. Immunostaining for CEACAM1 on sections of embedded HITA rings demonstrate the presence of CEACAM1-positive cells within the lumen (A) and in the outside of HITA rings (B). Mature endothelial cells lining HITA lumen and not involved in the sprouting remain negative for CEACAM1 (arrows). CD34 immunostaining of a serial section following the section in B confirms the involvement of CD34+ cells (arrows) in formation of these new capillaries at the border between vascular wall and the collagen gel (C). CEACAM1 is not present in the quiescent endothelial cells lining HITA lumen or in the vasculogenic zone of HITA when ring assay was not performed (D). (E-H) GFP marking of HITA-wall cells in ring assay. The injection of AdV5-GFP into a zone of the outer layer of the HITA demonstrates GFP-marked cells within and migrating out from the vascular wall (E). Higher magnification shows the sprouts from the vascular wall into the collagen gel (F). Focusing on another level demonstrates the formation of vascular like channel (arrows) at the border between the vascular wall and collagen gel corresponding to the vascular channels presented in B and C (G). No such sprouting is visible in the control ring without GFP-AdV5 (H).

View larger version (143K): [in a new window]   Fig. 5. Endothelial markers in cells sprouted from HITA wall during ring assay. Similar to that presented in the Fig. 4B cells isolated from the collagen gel after performing the ring assay also show a clear staining for CEACAM1 (A). Double immunostaining of these cells for CEACAM1 (dark) and VEGF receptor 2 (KDR) (red) demonstrate the colocalization of the VEGFR2 and CEACAM1 in the majority of the cells (B). Nearly all cells are also positive for the endothelial marker TIE2 (C). No specific staining is visible in the control section (D).

View larger version (186K): [in a new window]   Fig. 6. Capillary-like tube formation in vitro by cells sprouted from HITA rings. At different stages of confluence (A,B: non confluent; C,D: subconfluent), cells isolated from collagen gel after performing a ring assay formed a network of tubes (arrowheads) when stimulated with VEGF (A,C). No tubes were visible in controls exposed to basal medium only (B,D).

View larger version (51K): [in a new window]   Fig. 7. CD45 and CD68 positive cells in HITA wall before and after ring assay. Similar to CD34+ cells, CD45+ cells (arrows) are localized at the transitional zone between smooth muscle cell layer (SMC) and adventitial layer (ADV) (A, at higher magnification in B), but their number is significantly lower than that of CD34+ cells. No specific reaction is visible in endothelial cell layer (EC). Further immunohistochemical studies demonstrate in only a few cases sporadic and randomly distributed CD68+ cells in the adventitial layer (arrows) of untreated HITA (C). They are mostly localized distant from the mural zone where CD34+ and CD45+ cells were found. By contrast, a significant accumulation of CD68+ cells is visible in the adventitial layer including the zone containing the CD34+ and CD45+ cells when HITA rings were subjected to the ring assay (D). Some of these macrophages migrate into the collagen gel outside the ring (arrow) (E) and are involved in the capillary sprouts (arrow) in the collagen gel (arrow) (F). FACS-analyses of cells isolated from the collagen gel after ring assay demonstrate the existence of different cell populations such as CD133+ cells, CD45+ cells and CD105+ cells (G-I) among the cells migrated into the gel and/or involved in vessels sprouting from HITA. These FACS analyses show that approximately 20% of the CD133 positive cells exhibit CD45 (G). Unexpectedly, more than 60% of CD34+ cells co-express CD105 (H), although freshly isolated cells from the HITA wall did not express CD105 when prior to these analyses the pre-existing mature endothelial cells were removed via CD105 conjugated super paramagnetic microbeads. Furthermore, CD133+ cells also exhibit CD105 approximately in equal intensity (I). IgG-FITC and IgG-PE were used as control (J).

View larger version (110K): [in a new window]   Fig. 8. In vivo accumulation of macrophages in the vascular wall after arterial occlusion. (A,B) Post-mortem angiography shows the right femoral artery of a rat prior to (A) and 7 days after (B) its occlusion. The prominent appearance of collaterals (B, arrows) after femoral artery occlusion is seen, as reported earlier (Ito et al., 1997). (C,D) Immunofluorescence studies on sections from rat collateral arteries show KDR staining in endothelial cells (C) and the intramural accumulation of macrophages (ED2-antibody, red staining) 3 days after occlusion (D). No notable BrdU incorporation (E) is detectable in mature immunoselected macrophages from the wall of collateral arteries after femoral artery occlusion, indicating that these cells do not proliferate. CD133+ cells isolated from human mobilized peripheral blood and used as positive control show an extensive BrdU incorporation (F). DAPI staining was used to visualize all cells.

View larger version (99K): [in a new window]   Fig. 9. Recruitment of VW-EPC by tumor cells. HITA rings cocultured with cells of the human prostate cancer cell line DU-145 (A,B) show an extensive capillary outgrowth (arrows) into the lumen (A) and outside the rings (B) after 24 hours in culture. By contrast, no sprouts are seen in HITA rings cultured without tumor cells (C,D). Tumor cells (arrowheads) remain rounded and are not involved in the capillary outgrowth. Immunohistochemical studies on paraffin wax sections of several human organs show that CD34+ cells (arrows) are present in the wall of large and mid-sized arteries (E) and veins (F), as demonstrated here exemplarily on sections of human prostate. By contrast, CD31 immunostaining is only detectable in mature endothelial cells lining the lumina of arteries (G) and veins (H).

View larger version (37K): [in a new window]   Fig. 10. Hypothetical scheme of the `vasculogenic zone'. This scheme illustrates the concepts of the `vasculogenic zone', which is defined as a vascular mural zone containing EPCs and probably also multipotent mesodermal stem cells here. Although the CD34+/TIE2+/KDR+/VE-cadherin- cells present in this zone differentiate into endothelial cells and form capillary-like sprouts from the vascular wall, the multipotent stem cells in this zone may serve as precursors of the in vitro and in vivo observed accumulation of macrophages. Obviously, they also have the capacity to differentiate into smooth muscle cells.