Mesenchymal stem cells participating in ex vivo endothelium repair and its effect on vascular smooth muscle cells growth

BACKGROUND: Previous studies have shown that mesenchymal stem cells (MSCs) transplantation can promote neovascularization and regenerate damaged myocardium. However, it remains unknown whether MSCs seeding can be used to repair injured cellular components in vascular diseases. In this study we explored the feasibility of applying MSCs to endothelium repair in endothelial damage and vasoproliferative disorders. METHODS: Ex vivo model of endothelium repair was developed in which rabbit vascular smooth muscle cells (SMCs) were inoculated into the upper chamber and rabbit endothelial cells (ECs)/human MSCs into the lower chamber of a co-culture system. 3H-TdR incorporation and PCNA protein expression were assayed and migrated number of SMCs was calculated to evaluate the effect of MSCs seeding on SMCs growth. Flk-1 and vWF protein expressions were observed to analyze the plasticity of the seeded MSCs along endothelial lineage. RESULTS: In this co-culture system, no vWF protein but Flk-1 protein was observed in the 25.71% of MSCs after having been co-cultured with mature rabbit ECs for 5 days. Compared with the control group, the proliferation and migration of SMCs was significantly increased by proliferative ECs but decreased by confluent ECs (n=6, P<0.01). MSCs seeding decreased the proliferation and migration of SMCs compatible with the effect of proliferative ECs (n=6, P<0.001). However, no inhibition on SMCs growth was observed with MSCs seeding in comparison to the effect of confluent ECs. CONCLUSIONS: MSCs seeding can inhibit the proliferation and migration of SMCs. MSCs co-cultured with mature ECs have the ability to undergo milieu-dependent differentiation toward ECs.     

Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells

Angiogenesis is highly sensitive to the composition of the vascular microenvironment, however, our understanding of the structural and matricellular components of the vascular microenvironment that regulate angiogenesis and the molecular mechanisms by which these molecules function remains incomplete. Our previous results described a novel pro-angiogenic activity for Microfibril-Associated Glycoprotein-2 (MAGP-2), but did not address the molecular mechanism(s) by which this is accomplished. We now demonstrate that MAGP-2 promotes angiogenic cell sprouting by antagonizing Notch signaling pathways in endothelial cells. MAGP-2 decreased basal and Jagged1 induced expression from the Notch sensitive Hes-1 promoter in ECs, and blocked Jagged1 stimulated Notch1 receptor processing in transiently transfected 293T cells. Interestingly, inhibition of Notch signaling by MAGP-2 seems to be restricted to ECs since MAGP-2 increased Hes-1 promoter activity and Notch1 receptor processing in heterologous cell types. Importantly, constitutive activation of the Notch signaling pathway blocked the ability of MAGP-2 to promote angiogenic cell sprouting, as well as morphological changes associated with angiogenesis. Collectively, these observations indicate that MAGP-2 promotes angiogenic cell spouting in vitro by antagonizing Notch signaling pathways in ECs.     

Soluble HSPB1 regulates VEGF-mediated angiogenesis through their direct interaction

Endothelial cell function is critical for angiogenic balance in both physiological and pathological conditions, such as wound healing and cancer, respectively. We report here that soluble heat shock protein beta-1 (HSPB1) is released primarily from endothelial cells (ECs), and plays a key role in regulating angiogenic balance via direct interaction with vascular endothelial growth factor (VEGF). VEGF-mediated phosphorylation of intracellular HSPB1 inhibited the secretion of HSPB1 and their binding activity in ECs. Interestingly, co-culture of tumor ECs with tumor cells decreased HSPB1 secretion from tumor ECs, suggesting that inhibition of HSPB1 secretion allows VEGF to promote angiogenesis. Additionally, neutralization of HSPB1 in a primary mouse sarcoma model promoted tumor growth, indicating the anti-angiogenic role of soluble HSPB1. Overexpression of HSPB1 by HSPB1 adenovirus was sufficient to suppress lung metastases of CT26 colon carcinoma in vivo, while neutralization of HSPB1 promoted in vivo wound healing. While VEGF-induced regulation of angiogenesis has been studied extensively, these findings illustrate the key contribution of HSPB1-VEGF interactions in the balance between physiological and pathological angiogenesis.     

Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin

Infantile hemangiomas are composed of endothelial cells (ECs), endothelial progenitor cells (EPCs), as well as perivascular and hematopoietic cells. Our hypothesis is that hemangioma-derived EPCs (HemEPCs) differentiate into the mature ECs that comprise the major compartment of the tumor. To test this, we isolated EPCs (CD133(+)/Ulex europeus- I(+)) and mature ECs (CD133(-)/Ulex europeus-I(+)) from proliferating hemangiomas and used a previously described property of hemangioma-derived ECs (HemECs), enhanced migratory activity in response to the angiogenesis inhibitor endostatin, to determine if HemEPCs share this abnormal behavior. Umbilical cord blood-derived EPCs (cbEPCs) were analyzed in parallel as a normal control. Our results show that HemEPCs, HemECs, and cbEPCs exhibit increased adhesion, migration, and proliferation in response to endostatin. This angiogenic response to endostatin was consistently expressed by HemEPCs over several weeks in culture, whereas HemECs and cbEPCs shifted toward the mature endothelial response to endostatin. Similar mRNA-expression patterns among HemEPCs, HemECs, and cbEPCs, revealed by microarray analyses, provided further indication of an EPC phenotype. This is the first demonstration that human EPCs, isolated from blood or from a proliferating hemangioma, are stimulated by an angiogenesis inhibitor. These findings suggest that EPCs respond differently from mature ECs when exposed to angiogenic or antiangiogenic signals.     

Adult bone marrow-derived cells recruited during angiogenesis comprise precursors for periendothelial vascular mural cells

Bone marrow (BM)-derived cells are thought to participate in the growth of blood vessels during postnatal vascular regeneration and tumor growth, a process previously attributed to stem and precursor cells differentiating to endothelial cells. We used multichannel laser scanning confocal microscopy of whole-mounted tissues to study angiogenesis in chimeric mice created by reconstituting C57BL mice with genetically marked syngeneic BM. We show that BM-derived endothelial cells do not significantly contribute to tumor- or cytokine-induced neoangiogenesis. Instead, BM-derived periendothelial vascular mural cells were persistently detected at sites of tumor- or vascular endothelial growth factor-induced angiogenesis. Subpopulations of these cells expressed the pericyte-specific NG2 proteoglycan, or the hematopoietic markers CD11b and CD45, but did not detectably express the smooth muscle markers smooth muscle alpha-actin or desmin. Thus, the major contribution of the BM to angiogenic processes is not endothelial, but may come from progenitors for periendothelial vascular mural and hematopoietic effector cells.     

Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: insights from a novel human angiogenesis assay.

OBJECTIVES: An endothelial cell (EC)-specific angiogenesis assay was developed to functionally characterize angiogenic properties of 2 distinct putative endothelial progenitor cells (EPCs): early EPCs and late outgrowth endothelial cells (OECs). BACKGROUND: Endothelial progenitor cells promote revascularization of ischemic tissue. However, the nature of different EPCs and their role in angiogenesis remains debated. METHODS: Tubulogenesis was assessed by immunohistochemistry in co-cultures of differentiated ECs (including human umbilical vein, coronary artery, and microvascular ECs) or non-ECs with monolayers of human fibroblasts (MRC5). Using adaptations of the co-culture assay, early EPCs and OECs, isolated from peripheral blood mononuclear cells, were assessed by 3-dimensional immunofluorescence microscopy for their capacity for: 1) independent tubulogenesis; 2) incorporation into pre-existing vascular networks; and 3) paracrine angiogenic effects using transwell cultures. RESULTS: Branched interconnecting EC-specific tubules formed with all differentiated ECs after 72 h. Proangiogenic and antiangiogenic agents modulated tubulogenesis appropriately (vascular endothelial growth factor 10 ng: +142 +/- 13%, 1 microM anti-vascular endothelial growth factor: -44 +/- 7% vs. control, p < 0.001). In contrast, early EPCs, along with nonendothelial cell types, failed to independently form tubules or incorporate into differentiated EC tubules. Nevertheless, early EPCs indirectly augmented tubulogenesis by differentiated ECs even when physically separated by transwells (+115 +/- 4% vs. control; p < 0.001). By contrast, OECs independently formed tubules and incorporated into differentiated EC tubules but exerted no significant paracrine angiogenic effects. CONCLUSIONS: A novel EC-specific tubulogenesis assay highlights strikingly different angiogenic properties of different EPCs: late OECs directly participate in tubulogenesis, whereas early EPCs augment angiogenesis in a paracrine fashion, with implications for optimizing cell therapies for neovascularization.     

骨髓基质干细胞种植体外修复内皮及对血管平滑肌细胞增生的影响

目的探讨骨髓基质干细胞(MSCs)种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔血管内皮、平滑肌和人MSCs,通过细胞共培养模拟血管内皮修复过程,用流式细胞仪分析MSCs分子表型特征,免疫荧光细胞化学法观察与内皮共培养的MSCsFlk1和vWF蛋白表达,根据下室内皮生长状态及是否接种MSCs将其分为对照组、单纯MSCs组、融合内皮组、对数内皮组和MSCs种植组。氚胸腺嘧啶脱氧核苷(3HTdR)掺入检测平滑肌细胞DNA合成,Westernblot检测平滑肌细胞中增殖细胞核抗原蛋白表达。结果分离的MSCs表达基质细胞标志CD105和CD166,不表达造血干祖细胞和内皮细胞标志CD34、Flk1、vWF;与内皮共培养5天时,vWF染色仍为阴性,但约25.71%MSCs开始表达Flk1;MSCs种植组平滑肌细胞3HTdR掺入虽高于融合内皮组,但与对数内皮组比较显著降低;MSCs种植组平滑肌细胞PCNA蛋白吸光度相对值虽高于融合内皮组,但与对数内皮组比较明显减少。结论MSCs种植能抑制平滑肌细胞增生,种植在成熟内皮中的MSCs具有微环境依赖向内皮分化的能力。     

Hypoxia paradoxically inhibits the angiogenic response of isolated vessel explants while inducing overexpression of vascular endothelial growth factor

This study was designed to investigate how changes in O₂ levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1–4 % O₂. Additionally, aortic neovessels preformed in atmospheric O₂ lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O₂. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signaling molecules including VEGFR2, NRP1 and Prkc-beta while upregulating expression of VEGFR1. Medium conditioned by hypoxic cultures exhibited angiostatic and anti-VEGF activities likely mediated by sVEGFr1. Hypoxia reduced expression of VEGFR1 and VEGFR2 in endothelial cells while upregulating VEGFR1 in macrophages and VEGF in both macrophages and mural cells. Thus, changes in O₂ levels profoundly affect the endothelial response to angiogenic stimuli. These results suggest that hypoxia-induced angiogenesis is fine-tuned by complex regulatory mechanisms involving not only production of angiogenic factors including VEGF but also differential regulation of VEGFR expression in different cell types and production of inhibitors of VEGF function such as sVEGFR1.     

MCP-1 mediates TGF-beta-induced angiogenesis by stimulating vascular smooth muscle cell migration

Transforming growth factor-beta (TGF-beta) and its signaling mediators play crucial roles in vascular formation. Our previous microarray analysis identified monocyte chemoattractant protein-1 (MCP-1) as a TGF-beta target gene in endothelial cells (ECs). Here, we report that MCP-1 mediates the angiogenic effect of TGF-beta by recruiting vascular smooth muscle cells (VSMCs) and mesenchymal cells toward ECs. By using a chick chorioallantoic membrane assay, we show that TGF-beta promotes the formation of new blood vessels and this promotion is attenuated when MCP-1 activity is blocked by its neutralizing antibody. Wound healing and transwell assays established that MCP-1 functions as a chemoattractant to stimulate migration of VSMCs and mesenchymal 10T1/2 cells toward ECs. Furthermore, the conditioned media from TGF-beta-treated ECs stimulate VSMC migration, and inhibition of MCP-1 activity attenuates TGF-beta-induced VSMC migration toward ECs. Finally, we found that MCP-1 is a direct gene target of TGF-beta via Smad3/4. Taken together, our findings suggest that MCP-1 mediates TGF-beta-stimulated angiogenesis by enhancing migration of mural cells toward ECs and thus promoting the maturation of new blood vessels.     

Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth

The mechanisms by which bone marrow (BM)-derived stem cells might contribute to angiogenesis and the origin of neovascular endothelial cells (ECs) are controversial. Neovascular ECs have been proposed to originate from VEGF receptor 2-expressing (VEGFR-2+) stem cells mobilized from the BM by VEGF or tumors, and it is thought that angiogenesis and tumor growth may depend on such endothelial precursors or progenitors. We studied the mobilization of BM cells to circulation by inoculating mice with VEGF polypeptides, adenoviral vectors expressing VEGF, or tumors. We induced angiogenesis by syngeneic melanomas, APCmin adenomas, adenoviral VEGF delivery, or matrigel plugs in four different genetically tagged universal or endothelial cell-specific chimeric mouse models, and subsequently analyzed the contribution of BM-derived cells to endothelium in a wide range of time points. To study the existence of circulating ECs in a nonmyeloablative setting, pairs of genetically marked parabiotic mice with a shared anastomosed circulatory system were created. We did not observe specific mobilization of VEGFR-2+ cells to circulation by VEGF or tumors. During angiogenesis, abundant BM-derived perivascular cells were recruited close to blood vessel wall ECs but did not form part of the endothelium. No circulation-derived vascular ECs were observed in the parabiosis experiments. Our results show that no BM-derived VEGFR-2+ or other EC precursors contribute to vascular endothelium and that cancer growth does not require BM-derived endothelial progenitors. Endothelial differentiation is not a typical in vivo function of normal BM-derived stem cells in adults, and it has to be an extremely rare event if it occurs at all.     

Loss of bcl-2 during the senescence exacerbates the impaired angiogenic functions in endothelial cells by deteriorating the mitochondrial redox state

Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.     

Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells

To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34(+) hematopoietic progenitors at shear stresses of up to 2 dynes/cm(2). This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-alpha. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. Preincubation of HUVECs with anti-VCAM-1 or of MSCs with anti-VLA-4 antibodies suppressed binding of MSCs to HUVECs but did not enhance inhibition by anti-P-selectin, indicating that both P-selectin and VCAM-1 are equally required for this process. Intravital microscopy demonstrated the capacity of MSCs to roll and adhere to postcapillary venules in vivo in a mouse model in a P-selectin-dependent manner. Thus, MSCs interact in a coordinated fashion with ECs under shear flow, engaging P-selectin and VCAM-1/VLA-4.     

Cell-based and direct gene transfer-induced angiogenesis via a secreted chimeric fibroblast growth factor-1 (sp-FGF-1) in the chick chorioallantoic membrane (CAM)

Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor; its structure lacks a signal peptide for secretion. We previously reported that the overexpression of a secreted version of FGF-1 (sp-FGF-1) in microvascular endothelial cells (ECs) enhances cell migration [Partridge et al. J Cell Biochem 2000; 78(3): 487]. In the current study, we have examined the angiogenic effects of sp-FGF-1 in chicken chorioallantoic membranes (CAMs). Two methods of examining the effects of sp-FGF-1 in CAMs were used: cell-mediated transfection via bovine ECs and direct gene transfection. In the cell-mediated gene transfection, those eggs that were implanted with a gelatin sponge seeded with ECs stably transfected to over-express sp-FGF-1 protein showed a significant increase in angiogenesis inside the sponge when compared to eggs treated with vector control-transfected ECs. In the direct gene transfer, eggs received sp-FGF-1 showed a significant increase in vascularization when compared to eggs received vector alone plasmids. These CAM models are useful both for studying molecular mechanisms of angiogenesis and for developing better gene therapy strategies.     

Angiopoietins differentially influence in vitro angiogenesis by endothelial cells of different origin

Angiopoietins are important growth factors for vascular development and quiescence. They are promising targets for pro- or anti-angiogenic therapies in diverse pathologies, but the mechanisms of the ANGPT/TIE2 system are complex and not well understood. In the present study, the separate and combined effects of angiopoietin 1 and angiopoietin 2 were studied, using a recently developed in vitro angiogenesis model that allows both a quantitative and qualitative evaluation of the angiogenic cascade. This cell culture model was performed with microvascular endothelial cells (ECs) originating from different vascular beds, i.e. dermal ECs and cardiac ECs. In addition, the expression of the angiopoietins and the receptors, TIE1 and TIE2 was analyzed with RT-qPCR. This study revealed that the angiopoietins provoked a differential response in the two endothelial cultures. Both angiopoietin 1 as well as angiopoietin 2 elicited an angiogenic cascade in the dermal ECs but not in the cardiac ECs. In addition, the RT-qPCR data revealed marked differences in the endogenous expression pattern of these factors, indicating that the origin of endothelial cells might have an important impact on their angiogenic potential.     

VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells

Human CD133 (AC133)(+)CD34(+) stem and progenitor cells derived from fetal liver and from bone marrow and blood incorporate a functional population of circulating endothelial precursor cells. Vascular endothelial growth factor receptor 3 (VEGFR-3) regulates cardiovascular development and physiological and pathological lymphangiogenesis and angiogenesis. However, the origin of VEGFR-3(+) endothelial cells (ECs) and the mechanisms by which these cells contribute to postnatal physiological processes are not known, and the possible existence of VEGFR-3(+) lymphatic or vascular EC progenitors has not been studied. Using monoclonal antibodies to the extracellular domain of VEGFR-3, we show that 11% +/- 1% of CD34(+) cells isolated from human fetal liver, 1.9% +/- 0.8% CD34(+) cells from human cord blood, and 0.2% +/- 0.1% of CD34(+) cells from healthy adult blood donors are positive for VEGFR-3. CD34(+)VEGFR-3(+) cells from fetal liver coexpress the stem/precursor cell marker CD133 (AC133). Because mature ECs do not express CD133, coexpression of VEGFR-3 and CD133 on CD34(+) cells identifies a unique population of stem and progenitor cells. Incubation of isolated CD34(+)VEGFR-3(+) cells in EC growth medium resulted in a strong proliferation (40-fold in 2 weeks) of nonadherent VEGFR-3(+) cells. Plating of these cells resulted in the formation of adherent VEGFR-3(+)Ac-LDL(+) (Ac-LDL = acetylated low-density lipoprotein) EC monolayers expressing various vascular and lymphatic endothelial-specific surface markers, including CD34, VE-cadherin, CD51/61, CD105, LYVE-1, and podoplanin. These data demonstrate that human CD34(+)CD133(+) cells expressing VEGFR-3 constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal lymphangiogenesis and/or angiogenesis.     

Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors

The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24 h whereas TLR2, 4, and 8 expression peaks at 24 h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or mural cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial cell types by blocking mural cell recruitment and inducing formation of multinucleated giant cells by macrophages. TLR9-induced mural cell depletion is associated with reduced expression of the mural cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.     

New functions of the fibrinolytic system in bone marrow cell-derived angiogenesis

Angiogenesis is a process by which new blood vessels form from preexisting vasculature. This process includes differentiation of angioblasts into endothelial cells with the help of secreted angiogenic factors released from cells such as bone marrow (BM)-derived cells. The fibrinolytic factor plasmin, which is a serine protease, has been shown to promote endothelial cell migration either directly, by degrading matrix proteins such as fibrin, or indirectly, by converting matrix-bound angiogenic growth factors into a soluble form. Plasmin can also activate other pericellular proteases such as matrix metalloproteinases (MMPs). Recent studies indicate that plasmin can additionally alter cellular adhesion and migration. We showed that factors of the fibrinolytic pathway can recruit BM-derived hematopoietic cells into ischemic/hypoxic tissues by altering the activation status of MMPs. These BM-derived cells can function as accessory cells that promote angiogenesis by releasing angiogenic signals. This review will discuss recent data regarding the role of the fibrinolytic system in controlling myeloid cell-driven angiogenesis. We propose that plasmin/plasminogen may be a potential target not only for development of effective angiogenic therapeutic strategies for the treatment of cancer, but also for development of strategies to promote ischemic tissue regeneration.     

The thin prep rat aortic ring assay: a modified method for the characterization of angiogenesis in whole mounts

The rat aortic ring model has gained broad acceptance as an angiogenic assay. This system can be used to study the activity of angiogenic and anti-angiogenic factors, and investigate the molecular mechanisms of the angiogenic process. We describe here a thin prep modification of the aortic ring model, which significantly simplifies the procedure and allows staining of aortic outgrowths as whole mounts. Using this procedure, intact preparations of angiogenic outgrowths are successfully and reproducibly stained with endothelial cell (anti-CD-31 and -Tie2 antibodies, Griffonia Simplicifolia isolectin-B4) and smooth muscle cell (anti--smooth muscle actin antibody) markers. Combined use of double immunostaining and confocal microscopy allows concurrent visualization of endothelial and mural cells in the same cultures. Whole mount immunostains of rat aorta cultures are an effective way to rapidly characterize the cellular composition of the angiogenic outgrowths, and localize proteins implicated in the regulation of angiogenesis. This method should facilitate the work of the many vascular biologists that have adopted the rat aorta model as a tool to study angiogenesis and its mechanisms.