Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.     

Platelet-derived growth factor-BB (PDGF-BB) induces differentiation of bone marrow endothelial progenitor cell-derived cell line TR-BME2 into mural cells, and changes the phenotype

Blood vessels are composed of endothelial cells (EC) and mural cells, and the interaction between EC and mural cells is essential for the development and maintenance of the vasculature. EC differentiate from bone marrow-derived endothelial progenitor cells (EPC). Recently, we established a conditionally immortalized bone marrow EPC-derived cell line, TR-BME2, and a brain capillary EC (BCEC) line, TR-BBB, from temperature-sensitive-SV40 T-antigen gene transgenic rats. To understand the function of EPC, it is important to analyze the difference between EPC and mature EC such as BCEC. In this study, we identified EPC-specific genes by means of subtractive hybridization between TR-BME2 and TR-BBB. There was no significant difference between TR-BME2 and TR-BBB in the mRNA level of annexin II, which is expressed in EC. In contrast, the mRNA level of smooth muscle cell (SMC) markers such as smooth muscle protein 22 (SM22), calvasculin, and platelet-derived growth factor (PDGF) receptor-beta, was higher in TR-BME2 than in TR-BBB. Moreover, the mRNA level of contractile SMC markers, such as smooth muscle alpha-actin and SM22, was increased in the absence of EC growth factors, such as vascular endothelial growth factor. The mRNA level of synthetic SMC markers, such as matrix Gla protein, was increased by the addition of PDGF-BB. The SMC derived from TR-BME2 showed an altered phenotype, from contractile-type to synthetic-type, when they were cultured in the absence of PDGF-BB. These results show that TR-BME2 cells have higher levels of SMC markers compared with mature EC, and can differentiate into contractile- or synthetic-type SMC.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Liver cells isolated from pre‐clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody‐free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl₄ and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non‐parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen‐coated substrates. Isolated cells showed high viability (80%‐95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub‐populations from control or cirrhotic single livers without antibody selection.     

Adiponectin promotes endothelial cell differentiation from human peripheral CD14+ monocytes in vitro

Adiponectin was revealed to have anti-atherogenic and anti-inflammatory properties and has been recently found to stimulate angiogenesis in vivo and in vitro. However, the role of adiponectin in endothelial differentiation remains unclear. The objective of this study was to investigate whether adiponectin can promote peripheral CD14(+) monocytes differentiation into endothelial cells (ECs). Human peripheral blood CD14(+) monocytes were cultured with or without adiponectin (10 microg/ml) for 10 days. Adiponectin significantly promoted EC morphology formation from CD14(+) monocytes. By flow cytometery analysis, cells treated with adiponection substantially increased mean fluorescence intensity of vascular endothelial growth factor receptor-2 (VEGFR-2) and endothelial nitric oxide synthase (eNOS), two specific endothelial markers, by 49.2 % and 53.9 %, respectively, as compared to control cells. By real time PCR analysis, the mRNA level of eNOS in adiponectin-treated cells was also increased by 31.9 % of that of the control cells. However, the mRNA levels of calponin and SMMHC, two specific SMC markers, in adiponectin-treated cells were decreased by 81.1 % and 79.7 % of that of the control cells, respectively. These data demonstrated that adiponectin could promote endothelial differentiation from peripheral blood CD14(+) monocytes by morphology change, upregulation of EC markers and downregulation of SMC markers. Adiponectin-promoted EC differentiation may contribute to vascular healing and angiogenesis.     

Neovessel formation promotes liver fibrosis via providing latent transforming growth factor-β

Aim

Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-β, the precursor of the most fibrogenic cytokine TGF-β.

Methods

After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-β activation using primary cultured HSCs and liver sinusoidal ECs (LSECs).

Results

In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-β via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-β present in LSEC conditioned medium and subsequent HSC activation.

Conclusion

Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-β that activated on the surface of HSCs by PLK, thereby resultant active TGF-β stimulates the activation of HSCs.     

CD133+ hepatic stellate cells are progenitor cells

Hepatic stellate cells (HSC) play an important role in the development of liver fibrosis. Here, we report that HSC express the stem/progenitor cell marker CD133 and exhibit properties of progenitor cells. CD133+ HSC of rats were selected by specific antibodies and magnetic cell sorting. Selected cells displayed typical markers of HSC, endothelial progenitor cells (EPC), and monocytes. In cell culture, CD133+ HSC transformed into alpha-smooth muscle actin positive myofibroblast-like cells, whereas application of cytokines known to facilitate EPC differentiation into endothelial cells led to the formation of branched tube-like structures and induced expression of the endothelial cell markers endothelial nitric oxide synthase and vascular-endothelial cadherin. Moreover, cytokines that guide stem cells to develop hepatocytes led to the appearance of rotund cells and expression of the hepatocyte markers alpha-fetoprotein and albumin. It is concluded that CD133+ HSC are a not yet recognized progenitor cell compartment with characteristics of early EPC. Their potential to differentiate into endothelial or hepatocyte lineages suggests important functions of CD133+ HSC during liver regeneration.     

Absence of OX-43 antigen expression in invasive capillary sprouts: identification of a capillary sprout-specific endothelial phenotype

Endothelial cells exhibit a number of unique phenotypes, some of which are angiogenesis dependent. To identify a capillary sprout-specific endothelial phenotype, we labeled angiogenic rat mesentery tissue using a microvessel and capillary sprout marker (laminin), selected endothelial cell markers (CD31, tie-2, and BS-I lectin), and the OX-43 monoclonal antibody, which recognizes a 90-kDa membrane glycoprotein of unknown function. In tissues that were stimulated through wound healing and compound 48/80 application, double-immunolabeling experiments with an anti-laminin antibody revealed that the OX-43 antigen was expressed strongly in all microvessels. However, the OX-43 antigen was completely absent from a large percentage (>85%) of the capillary sprouts that were invading the avascular tissue space. In contrast, sprouts that were introverting back into the previously vascularized tissue retained high levels of OX-43 antigen expression. Double-labeling experiments with endothelial markers indicated that the OX-43 antigen was expressed by microvessel endothelium but was absent from virtually all invasive capillary sprout endothelial cells. We conclude that the absence of OX-43 antigen expression marks a novel, capillary sprout-specific, endothelial cell phenotype. Endothelial cells of this phenotype are particularly abundant in capillary sprouts that invade avascular tissue during angiogenesis.     

Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis

Focal adhesion kinase (FAK) is a critical mediator of signal transduction by integrins and growth factor receptors in a variety of cells including endothelial cells (ECs). Here, we describe EC-specific knockout of FAK using a Cre-loxP approach. In contrast to the total FAK knockout, deletion of FAK specifically in ECs did not affect early embryonic development including normal vasculogenesis. However, in late embryogenesis, FAK deletion in the ECs led to defective angiogenesis in the embryos, yolk sac, and placenta, impaired vasculature and associated hemorrhage, edema, and developmental delay, and late embryonic lethal phenotype. Histologically, ECs and blood vessels in the mutant embryos present a disorganized, detached, and apoptotic appearance. Consistent with these phenotypes, deletion of FAK in ECs isolated from the floxed FAK mice led to reduced tubulogenesis, cell survival, proliferation, and migration in vitro. Together, these results strongly suggest a role of FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.     

Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells.     

Liver sinusoidal endothelial cells are insufficient to activate T cells

Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45(-)) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and FcgammaRs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4(+) or CD8(+) TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.     

Notch1 activation in embryonic VE-cadherin populations selectively blocks hematopoietic stem cell generation and fetal liver hematopoiesis

Hematopoietic stem cells (HSC) are found in several independent sites embryonically. Loss-of-function studies indicated that Notch1, but not Notch2 signaling was required for HSC emergence from the aortic-gonado-mesonephros (AGM) region. We previously showed that constitutive Notch1 activation impaired primitive erythroid differentiation, but its effects on HSC emergence from the AGM region were not studied. To further define specific roles of Notch receptors, we characterized HSC in mouse embryos expressing either Notch1 intracellular domain (ICD) or Notch4ICD in VE-cadherin or SM22α expressing populations. Although embryonic Notch1 activation in VE-cadherin populations led to lethality after E13.5, earlier defects in the fetal liver were observed. Embryos were analyzed at E12.5 to assess hematopoiesis and the phenotype of developing cells in the AGM region. We found that activation of Notch1 in the endothelial compartment in VE-cadherin expressing cells resulted in the absence of intra-aortic clusters and defects in fetal liver hematopoiesis. In contrast, although Notch4 expression is regulated during fetal hematopoiesis, activation of Notch4 in VE-cadherin expressing populations did not affect HSC phenotype, although later vascular remodeling was impaired. Likewise, activation of Notch1 in SM22α positive populations had no significant effect on hematopoiesis. Our results indicate a cell type-dependent activity and distinct features of Notch1 versus Notch4 signaling and their impact on HSC generation.     

Chemotherapeutic stress selectively activates NF-kappa B-dependent AKT and VEGF expression in liver cancer-derived endothelial cells

Targeting endothelial cells (EC) that line tumor blood vessels forms the basis for metronomic therapy and is a promising new strategy for the treatment of cancer. Genetic and phenotypic differences between tumor-derived and normal ECs indicate that targeting tumor ECs may be therapeutically useful. In the present study, we examined differences in responses to chemotherapy in microvascular EC lines from tumoral (T-EC) and normal (N-EC) mouse liver tissues. The identity of these cells was confirmed by immunocytochemistry for EC markers, such as vascular endothelial-cadherin and CD31 for both types of ECs, and the tumor-endothelial-specific marker tumor endothelial marker-7 for T-EC. The involvement of Akt in NF-kappaB-dependent angiogenesis was different between N-EC and T-EC. Chemotherapeutic stress increased angiogenesis in T-EC, but not N-EC via an NF-kappaB-Akt-dependent manner. Both NF-kappaB and Akt were involved in enhanced survival and migration in T-EC in response to chemotherapeutic stress. Moreover, Akt was involved in NF-kappaB-dependent VEGF expression and angiogenesis. These studies, showing differences in cellular responses to chemotherapy in tumor-derived ECs, indicate that specific therapies targeting these cells may be therapeutically useful for liver cancers.     

Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell-cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum-free ECCM with antiplatelet-derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF-like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.     

Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine,MCP-1 and activation of TLR4 and Gi/o proteins signaling

The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.     

Proteomics analysis of plasma membrane from liver sinusoidal endothelial cells after partial hepatectomy by an improved two-dimensional electrophoresis

Liver regeneration is an angiogenesis-associated phenomenon. To identify key plasma membrane (PM) proteins of endothelial cells involved in the initiation of angiogenesis during liver regeneration, the PM of liver sinusoidal endothelial cells (LSEC) at 72 h after partial hepatectomy was enriched by an established in vivo membrane density perturbation method. The differentially expressed membrane proteins compared to those from sham operation were quantified using an improved two-dimensional 16-BAC/SDS-PAGE and identified by LC-MS/MS. Several proteins were further confirmed by cICAT labeling quantitative strategy. A total of 47 proteins were identified including known and novel proteins involved in angiogenesis or liver regeneration, such as inducible nitric oxide synthase, type IV collagen, and integrin beta3. Our results indicated that the combination of the membrane density perturbation strategy and the improved two-dimensional electrophoresis (2-DE) method are useful for investigating the endothelial dysfunctions in vivo.     

UC blood hematopoietic stem cells and therapeutic angiogenesis

Studies suggest that mobilized hematopoietic stem cells (HSC) are recruited to ischemic tissue and stimulate angiogenesis. Critical observations in pre-clinical studies have identified an augmentation of endogenous microvascular collateralization that is beyond that directly attributable to anatomic incorporation and differentiation of infused human cells into the vascular endothelium. Evidence links age-associated reductions in the levels of circulating marrow-derived HSC characterized by expression of early HSC markers CD133 and CD34, with the occurrence of cardiovascular events and associated death. Utilizing the patient's own HSC to augment angiogenesis has several disadvantages, including reduced function of these cells and logistical issues related to cell collection from individual patients. Thus an available source of allogeneic HSC such as UC blood for cellular therapy may be optimal.