Degradation of soluble VEGF receptor-1 by MMP-7 allows VEGF access to endothelial cells

Vascular endothelial growth factor (VEGF) signaling in endothelial cells serves a critical role in physiologic and pathologic angiogenesis. Endothelial cells secrete soluble VEGF receptor-1 (sVEGFR-1/sFlt-1), an endogenous VEGF inhibitor that sequesters VEGF and blocks its access to VEGF receptors. This raises the question of how VEGF passes through this endogenous VEGF trap to reach its membrane receptors on endothelial cells, a step required for VEGF-driven angiogenesis. Here, we show that matrix metalloproteinase-7 (MMP-7) degrades human sVEGFR-1, which increases VEGF bioavailability around the endothelial cells. Using a tube formation assay, migration assay, and coimmunoprecipitation assay with human umbilical vein endothelial cells (HUVECs), we show that the degradation of sVEGFR-1 by MMP-7 liberates the VEGF(165) isoform from sVEGFR-1. The presence of MMP-7 abrogates the inhibitory effect of sVEGFR-1 on VEGF-induced phosphorylation of VEGF receptor-2 on HUVECs. These data suggest that VEGF escapes the sequestration by endothelial sVEGFR-1 and promotes angiogenesis in the presence of MMP-7.     

Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro

Patients with myocardial infarction reveal an altered number of circulating mesenchymal stem cells (MSCs). Recently, it was shown that MSCs are able to regenerate myocardial tissue and to differentiate into endothelial cells. The homing mechanisms of MSCs from the circulation into the target tissue, however, are not understood so far. In this study, we evaluated the impact of platelets on MSC recruitment, proliferation, migration and integration into the endothelium. MSCs expressing α_vβ₃ integrin were recruited to human arterial endothelial cells exposed to isolated platelets or IL-1β under high shear conditions. Furthermore, induction of vascular injury in vivo resulted in increased recruitment of injected MSCs as assessed by intravital microscopy and depletion of platelets significantly reduced this adhesion. The interaction of platelets and MSCs was inhibited by pre-incubation with the mAb 7E3 or an RGD protein both blocking β₃ integrin mediated adhesion. Platelets had a chemotactic effect on MSCs, promoted a migratory MSC phenotype and dose- and activation-dependently enhanced migration of MSCs, a process, which was mediated by basic fibroblast growth factor (bFGF). Similarly, platelet derived bFGF increased proliferation of MSCs. Coincubation of MSCs with platelets facilitated integration into an endothelial monolayer, which was significantly reduced by pre-incubation with a blocking mAb to bFGF. We conclude that platelets may play a critical part in the recruitment of MSCs to the endothelium, influence MSC function and promote integration of MSCs into the endothelium.     

VASH2对脑胶质瘤血管新生的影响机制

目的 通过观察血管抑制素-2(VASH2)对脑胶质瘤微血管内皮细胞增殖、迁移和管形成的调控作用,探究其对脑胶质瘤血管新生的影响机制.方法 构建人脑胶质瘤微血管内皮细胞模型,通过稳定转染获得VASH2过表达和表达沉默的细胞系,在后续实验中分组,分别为空白对照组、VASH2过表达阴性对照空质粒组[VASH2(+)NC组]、...     

Signaling regulation of fetoplacental angiogenesis

During normal pregnancy, dramatically increased placental blood flow is critical for fetal growth and survival as well as neonatal birth weights and survivability. This increased blood flow results from angiogenesis, vasodilatation, and vascular remodeling. Locally produced growth factors including fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are key regulators of placental endothelial functions including cell proliferation, migration, and vasodilatation. However, the precise signaling mechanisms underlying such regulation in fetoplacental endothelium are less well defined, specifically with regard to the interactions amongst protein kinases (PKs), protein phosphatase, and nitric oxide (NO). Recently, we and other researchers have obtained solid evidence showing that different signaling mechanisms participate in FGF2- and VEGFA-regulated fetoplacental endothelial cell proliferation and migration as well as NO production. This review will briefly summarize currently available data on signaling mediating fetoplacental angiogenesis with a specific emphasis on PKs, ERK1/2, AKT1, and p38 MAPK and protein phosphatases, PPP2 and PPP3.     

Lysophosphatidic acid mediates interleukin-8 expression in human endometrial stromal cells through its receptor and nuclear factor-kappaB-dependent pathway: a possible role in angiogenesis of endometrium and placenta

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid molecule involved in inflammation, angiogenesis, would healing, and cancer invasion. Whereas serum lysophospholipase D activity increases in women with pregnancy, the role of LPA in pregnancy remains unclear. We investigated the expression of LPA receptors and function of LPA in endometrial stromal cells. Histologically normal endometrium was obtained from surgical specimens of women undergoing hysterectomy for leiomyoma. First-trimester decidua was obtained from women receiving elective termination of pregnancy. We examined the expressions of LPA1, LPA2, and LPA3 receptors in endometrial stromal cells. The effects of LPA on the expression of vascular endothelial growth factor, IL-6, and IL-8 were examined. Signal pathways of LPA were delineated. Functions of secretory angiogenic factors were tested using human endometrial microvascular endothelial cells. Immunoreactivity and mRNA of LPA1 receptors were identified in endometrial stromal cells. LPA enhanced IL-8 expression in a dose- and time-dependent manner, whereas vascular endothelial growth factor or IL-6 expression was not affected by LPA treatment. Mechanistic dissection disclosed that LPA functioned via the Gi protein, MAPK/p38 and nuclear factor-kappaB pathway. LPA-induced IL-8 enhanced migration, permeability, capillary tube formation, and proliferation of human endometrial microvascular endothelial cells. Endometrial stromal cells express LPA1 receptors. Through the LPA1 receptor, LPA induces IL-8 expression via a nuclear factor-kappaB-dependent signal pathway. These results could suggest that LPA may play a role in angiogenesis of endometrium and placenta through induction of IL-8 in endometrial stromal cells during pregnancy.     

Involvement of E-selectin in recruitment of endothelial progenitor cells and angiogenesis in ischemic muscle

E-selectin plays critical roles in tethering leukocytes to endothelial cells (ECs). We studied the role of E-selectin in endothelial progenitor cell (EPC) homing and vasculogenesis. After ischemia, the expression of E-selectin on ECs peaked 6 to 12 hours and returned to baseline at 24 hours, whereas the level of soluble E-selectin (sE-selectin) in serum increased over 24 hours and remained high at day 7. Mouse bone marrow-derived EPCs expressed not only E-selectin but also its ligand. Homing of circulating EPCs to ischemic limb was significantly impaired in E-selectin knock-out mice, as well as wild-type mice pretreated with blocking antibody against E-selectin, which was rescued by local sE-selectin injection. Mechanism for this is that sE-selectin stimulated not only ECs to express ICAM-1, but also EPCs to secrete interleukin-8 (IL-8), leading to enhanced migration and incorporation to ECs capillary formation. In therapeutic aspect, local treatment with sE-selectin enhanced efficacy of EPC transplantation for vasculogenesis and salvage of ischemic limb. Conversely, when E-selectin was knocked down by E-selectin small interfering RNA, blood flow recovery after EPC transplantation was significantly impaired. But this impaired vasculogenesis was rescued by sE-selectin. In conclusion, these data demonstrate E-selectin is a pivotal molecule for EPCs' homing to ischemic limb and vasculogenesis.     

Targeted migration of mesenchymal stem cells modified with CXCR4 to acute failing liver improves liver regeneration

AIM: To improve the colonization rate of transplanted mesenchymal stem cells (MSCs) in the liver and effect of MSC transplantation for acute liver failure (ALF).METHODS: MSC was modified with the chemokine CXC receptor 4 (CXCR4) gene (CXCR4-MSC) or not (Null-MSC) through lentiviral transduction. The characteristics of CXCR4-MSCs and Null-MSCs were determined by real-time quantitative polymerase chain reaction, Western blotting and flow cytometry. CXCR4-MSCs and Null-MSCs were infused intravenously 24 h after administration of CCl₄ in nude mice. The distribution of the MSCs, survival rates, liver function, hepatocyte regeneration and growth factors of the recipient mice were analyzed.RESULTS: In vitro, CXCR4-MSCs showed better migration capability toward stromal cell-derived factor-1α and a protective effect against thioacetamide in hepatocytes. In vivo imaging showed that CXCR4-MSCs migrated to the liver in larger numbers than Null-MSCs 1 and 5 d after ALF. Higher colonization led to a longer lifetime and better liver function. Either CXCR4-MSCs or Null-MSCs exhibited a paracrine effect through secreting hepatocyte growth factor and vascular endothelial growth factor. Immunohistochemical analysis of Ki-67 showed increased cell proliferation in the damaged liver of CXCR4-MSC-treated animals.CONCLUSION: Genetically modified MSCs expressing CXCR4 showed greater colonization and conferred better functional recovery in damaged liver.     

Oncostatin M induces angiogenesis and cartilage degradation in rheumatoid arthritis synovial tissue and human cartilage cocultures

OBJECTIVE: To investigate the role of oncostatin M (OSM) in cell adhesion, angiogenesis, and matrix degradation in rheumatoid arthritis (RA) synovial tissue and normal human cartilage. METHODS: Human dermal microvascular endothelial cell (HDMEC) and RA synovial fibroblast (RASF) proliferation and intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression were assessed by a bromodeoxyuridine proliferation assay and flow cytometry. HDMEC tubule formation and migration were assessed by Matrigel culture and migration assay. Production of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in RA synovial explants, and proteoglycan/glycosaminoglycan (GAG) release, vascular endothelial growth factor (VEGF), and angiopoietin 2 production from RASF/normal cartilage cocultures were assessed by enzyme-linked immunosorbent assay and immunohistology. RESULTS: HDMEC/RASF proliferation was induced by OSM and interleukin-1beta (IL-1beta), alone and in combination. OSM enhanced cell surface expression of ICAM-1, but not VCAM-1, on endothelial cells and RASFs. OSM increased endothelial cell tubule formation and migration. In RA synovial explants, OSM induced production of MMP-1 and TIMP-1. When OSM was combined with IL-1beta, however, the MMP-1:TIMP-1 ratio was significantly increased. OSM potentiated IL-1beta-induced MMP-1 and MMP-13 expression in normal human cartilage/RASF cocultures, resulting in a significant increase in the MMP:TIMP ratio. In OSM/IL-1beta- stimulated cocultures, cartilage sections demonstrated significant proteoglycan depletion that was paralleled by a significant increase in GAG release in supernatants. Finally, compared with either cytokine alone, the combination of OSM and IL-1beta significantly induced VEGF production in RASF/cartilage cocultures. CONCLUSION: These data suggest that OSM promotes angiogenesis and endothelial cell migration and potentiates the effects of IL-1beta in promoting extracellular matrix turnover and human cartilage degradation. Furthermore, the induction of VEGF in cocultures supports the hypothesis of a link between angiogenesis and cartilage degradation.     

Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes

A replication-defective recombinant retrovirus containing the human papilloma virus E6/E7 genes (LXSN-16 E6E7) was used to immortalize stromal cells from human marrow. The E6/E7 gene products interfere with the function of tumor-suppressor proteins p53 and Rb, respectively, thereby preventing cell cycle arrest without causing significant transformation. Twenty-seven immortalized clones designated HS-1 to HS- 27 were isolated, four of which are characterized in this report. Two cell lines, HS-5 and HS-21, appear to be fibroblastoid and secrete significant levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), Kit ligand (KL), macrophage-inhibitory protein-1 alpha, interleukin-6 (IL-6), IL- 8, and IL-11. However, only HS-5 supports proliferation of hematopoietic progenitor cells when cocultured in serum-deprived media with no exogenous factors. Conditioned media (CM) from HS-5 promotes growth of myeloid colonies to significantly greater extent than a cocktail of recombinant factors containing 10 ng/mL of IL-1, IL-3, IL- 6, G-CSF, GM-CSF, and KL and 3 U of erythropoietin (Epo). Two additional clones, HS-23 and HS-27, resemble "blanket" cells, with an epithelioid morphology, and are much larger, broader, and flatter when compared with HS-5 and HS-21. These lines secrete low levels of growth factors and do not support proliferation of isolated progenitor cells in cocultures. CM from HS-23 and HS-27 also fail to support growth of myeloid colonies. Both HS-23 and HS-27 express relatively high levels of VCAM-1, yet HS-27 is the only line that supports the formation of "cobblestone" areas by isolated CD34+38lo cells. We hypothesize that HS- 5, HS-21, HS-23, and HS-27 represent functionally distinct components of the marrow microenvironment.     

Stimulation of tube formation mediated through the prostaglandin EP2 receptor in rat luteal endothelial cells

To explore the role of prostaglandin E(2) (PGE(2)) in angiogenesis in the developing corpus luteum, luteal microvascular endothelial-like cells (luteal ECs) were prepared from highly luteinizing ovaries of rats using the percoll density gradient method. The cells abundantly expressed the mRNAs of the endothelial markers CD31 (PECAM-1) and responded to the vascular endothelial growth factor (VEGF) to form in vitro tube structures on Matrigel. Cyclooxygenase (COX) inhibitors significantly suppressed tube formation in luteal ECs, whereas PGE(2) counteracted the COX inhibitor-induced blockage. PGE(2)-induced tube formation was blocked by a cyclic AMP-dependent protein kinase A (PKA) inhibitor, H89. The antagonist against the PGE receptor type 2 (EP2 receptor), AH6809, completely inhibited PGE(2)-induced tube formation and partly suppressed the VEGF-induced tube formation but did not attenuate PGE(2)-induced phosphorylation of both AKT kinase and extracellular signal-regulated kinase 1/2. VEGF significantly enhanced the expression of COX-2 mRNAs detected by real-time RT-PCR and PGE(2) secretion into the media measured by ELISA in luteal ECs. In turn, PGE(2) stimulated VEGF expression. In vitro co-culture of luteal ECs with steroidogenic luteal cells (SLCs) promoted tube formation. Pre-treatment of SLCs with VEGF further enhanced tube formation of ECs, and this effect was blocked by the COX-2 inhibitor. This stimulatory effect was inhibited by treatment with AH6809. These data indicate that PGE(2) exerts a direct stimulatory effect on tube formation mainly via the EP2 receptor/PKA signaling in luteal ECs. Our results suggest the possibility that the endogenous PGE(2) that is produced from luteinizing follicular cells as well as ECs may stimulate luteal angiogenesis.     

Potential active compounds and molecular mechanism of Xuefu Zhuyu decoction for atherosclerosis,based on network pharmacology and molecular docking

To explore the potential active compounds and molecular mechanism of Xuefu Zhuyu decoction (XFZYD) in the treatment of atherosclerosis (AS) based on network pharmacology and molecular docking.The effective components and action targets of XFZYD were screened by using TCMSP database. And then, the action targets of AS were collected by GeneCards database. The intersection targets between the effective components’ targets of XFZYD and AS-related action targets were used to construct PPI networks. GO and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed on these intersection targets. Finally, molecular docking software was used to excavate the active compounds of the core targets VEGFA and AKT1.We detected 225 active components of XFZYD, and found that quercetin, kaempferol, luteolin, naringenin, β-sitosterol, isorhamnetin, stigmasterol, baicalein, nobiletin, and β-carotene are the potential active compounds of XFZYD; STAT3, IL6, JUN, VEGFA, MAPK14, and AKT1 are the core target proteins of the active compounds, among which VEGFA and AKT1 are the key target proteins. PPI network results showed that β-carotene, quercetin, kaempferol, luteolin, and naringenin had higher degree values and more corresponding targets than other 5 active compounds and had the stable binding ability to regulatory proteins VEGFA and AKT1. The core components β-carotene, quercetin, kaempferol, and luteolin exerted their therapeutic effects on AS by acting on the key target proteins VEGFA and AKT1 to regulate fluid shear stress and the AGE-RAGE signaling pathway and IL-17 signaling pathway of diabetic complications of AS. The molecular docking results showed that VEGFA and AKT1 had great docking ability with the targeted active compounds, and β-carotene is the best.The active components of XFZYD, including β -carotene, quercetin, kamanol, and luteolin, can act on VEGFA and AKT1. These active ingredients play a role in alleviating and treating AS by regulating fluid shear stress and participating in signaling pathways such AS AGE-RAGE of atherosclerosis and diabetes mellitus complicated with AS. β-carotene is a potential inhibitor of VEGFA and AKT1 and treats AS through antioxidant action.     

Redox signaling in angiogenesis: role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.     

Conditioned medium from contracting skeletal muscle cells reverses insulin resistance and dysfunction of endothelial cells

ObjectiveObese adipose tissue has been characterized with chronic inflammation associated with elevated secretion of inflammatory cytokines and declined secretion of anti-inflammatory cytokines which can impair endothelial function in an endocrine manner. Adipose tissue hypoxia plays a role in the changes of cytokines. Physical exercise/muscle contraction may help preventing cardiovascular disease through improving insulin resistance and endothelium function. However the mechanism is unclear. Skeletal muscle is an endocrine tissue. Contracting muscles secrete myokines which may play roles in the beneficial effect of exercise. In this study, the conditioned medium from electrical pulse stimulation (EPS) regulated skeletal muscle cells was used to explore the mechanism of contraction on endothelial dysfunction and insulin resistance induced by conditioned medium from hypoxic adipocytes.Methods3T3-L1 adipocytes were incubated under normoxia or hypoxia condition, respectively. The supernatant was collected as adipocyte conditioned medium (CM-N and CM-H). C2C12 mouse skeletal muscle cells were stimulated with EPS for 12 h. The supernatant was collected as muscle cells conditioned medium (CM-EPS). Human umbilical vein endothelial cells (HUVECs) were incubated with adipocyte CM and muscle cells CM together. Macrophages migration to HUVECs was detected with transwell system. The mRNA expressions of E-selectin, ICAM-1, MCP-1 and IL-6 were measured by real-time PCR. The phosphorylation of IKKα/β, NF-κB, Akt, AMPK, eNOS and SOCS3 protein levels were detected by Western blot. Concentration of NO was measured by ELISA kit. HUVECs apoptosis was detected by flow cytometry.ResultsCM-EPS reduced the increase of mRNA expressions of E-selectin, ICAM-1, MCP-1 and IL-6 in HUVECs induced by CN-H. The phosphorylations of IKKα/β and NF-κB, SOCS3 protein level and endothelial cells apoptosis, which were raised by CM-H, were significantly reduced by CM-EPS. CM-EPS reversed the effects of CM-H on Akt and eNOS phosphorylations and NO production in HUVECs. CM-EPS directly stimulated the phosphorylation of AMPK, which caused the following phosphorylation of eNOS in HUVECs.ConclusionIn summary, CM-EPS reversed endothelial cells inflammation, apoptosis, insulin resistance and dysfunction induced by CM-H.     

槲皮素对食管癌Eca109细胞迁移侵袭及血管生成的影响

目的研究槲皮素(Que)对人食管癌Eca109细胞迁移侵袭及血管生成的影响。方法分别用5μg/mL、10μg/mL Que处理Eca109细胞,通过平板克隆形成实验、创伤愈合实验及Transwell实验观察其克隆形成能力以及迁移和侵袭能力的改变。制备Eca109肿瘤条件培养基,诱导人脐静脉内皮细胞CRL-1730迁移及成管,观察Que对其的影响。采用蛋白质免疫印迹法(Western blot)测定Que对Eca109细胞的血管内皮生长因子-A(VEGF-A)、基质金属蛋白酶2(MMP2)、MMP9蛋白表达的影响。结果 10μg/mL Que可显著抑制Eca109细胞的单细胞克隆形成能力(P0.05),而5μg/mL Que则不影响Eca109细胞的单细胞克隆形成能力(P0.05)。10μg/mL Que可抑制Eca109细胞的迁移和侵袭(P0.05),5μg/mL Que仅抑制Eca109细胞的侵袭(P0.05),但并不明显影响其迁移(P0.05)。5μg/mL、10μg/mL Que均可抑制Eca109肿瘤条件培养基诱导的CRL-1730细胞迁移和管腔形成(P均0.05),且10μg/mL Que的效果更明显。10μg/mL Que可明显降低VEGF-A、MMP2和MMP9的表达(P均0.05),5μg/mL Que仅降低MMP2的表达(P0.05)。结论 Que可以抑制Eca109细胞的迁移、侵袭及血管新生,并呈剂量依赖性。     

Proteasome Inhibitor MG132 Inhibits Angiogenesis in Pancreatic Cancer by Blocking NF-κB Activity

Since angiogenesis enables solid tumors, including pancreatic cancer (PaCa), to grow and metastasize, the development of anti-angiogenic agents is currently one of the urgent issues. Proteasome inhibitors are well known for inhibiting nuclear factor-kappa B (NF-κB) activity in various cancer cells, but little is known about their biologic mechanisms against angiogenesis in PaCa. We divided human PaCa cell lines into high-angiogenic (BxPC-3 and SW 1990) and low-angiogenic (MIA PaCa-2 and Capan-2) groups. The high-angiogenic PaCa cell lines constitutively expressed high NF-κB activity and produced high levels of vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8). The conditioned media from BxPC-3 significantly enhanced both proliferation of and tube formation by human umbilical vein endothelial cells (HUVECs) and these enhancements were significantly inhibited by the proteasome inhibitor MG132 treatment. Collectively, MG132 blocked PaCa-derived VEGF and IL-8 production through inhibition of NF-κB activity. Thus, proteasome inhibitors may prove beneficial as anti-angiogenic therapy for PaCa. Our studies show that MG132, a proteasome inhibitor, significantly blocked pancreatic-cancer-associated angiogenesis through inhibition of NF-κB and NF-κB-dependent proangiogenic gene products VEGF and IL-8.     

Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells: new insight into the pathogenesis of systemic sclerosis

OBJECTIVE: Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair. METHODS: MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony-forming unit-fibroblastoid colonies was determined. After culture in endothelial-specific medium, the endothelial-like MSC (EL-MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed. RESULTS: MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR-2+, CXCR4+, VEGFR-2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL-MSCs showed increased expression of VEGFR-1, VEGFR-2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell-derived factor 1 to cultured SSc EL-MSCs increased their angiogenic potential less than that in controls. CONCLUSION: Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.     

Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells

Circulating endothelial progenitor cells (EPC) are incorporated into newly formed capillaries, enhance neovascularization after hind limb ischemia and improve cardiac function after ischemic injury. Incorporated progenitor cells may also promote neovascularization and cardiac regeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilize tissue residing progenitor cells. Therefore, we analyzed the expression profile of cytokines in human peripheral blood-derived EPC as opposed to human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and CD14(+) monocytes by microarray technology. A gene tree analysis revealed a distinct expression pattern of angiogenic growth factors in EPC, mature endothelial cells, and CD14(+) monocytes. VEGF-A, VEGF-B, SDF-1, and IGF-1 mRNA levels were higher in EPC as compared to HUVEC or HMVEC. The enhanced mRNA expression was paralleled by a significant release of VEGF, SDF-1, and IGF-1 protein into the cell culture supernatant of EPC. Moreover, immunohistological analysis of ischemic limbs from nude rats revealed that VEGF is also released from recruited human EPC in vivo. As a functional consequence, conditioned medium of EPC induced a strong migratory response of mature endothelial cells, which was significantly inhibited by VEGF and SDF-1 neutralizing antibodies. Finally, conditioned medium of EPC significantly stimulated the migration of cardiac resident c-kit(+) progenitor cells in vitro. Taken together, EPC exhibit a high expression of angiogenic growth factors, which enhanced migration of mature endothelial cells and tissue resident cardiac progenitor cells. In addition to the physical contribution of EPC to newly formed vessels, the enhanced expression of cytokines may be a supportive mechanism to improve blood vessel formation and cardiac regeneration after cell therapy.     

PGE2 promotes angiogenesis through EP4 and PKA Cγ pathway

Inflammation is increasingly recognized as a critical mediator of angiogenesis, and unregulated angiogenic response is involved in human diseases, including cancer. Proinflammatory prostaglandin E2 (PGE2) is secreted by many cell types and plays important roles in the process of angiogenesis via activation of cognate EP1-4 receptors. Here, we provide evidence that PGE2 promotes the in vitro tube formation of human microvascular endothelial cells, ex vivo vessel outgrowth of aortic rings, and actual in vivo angiogenesis. Use of EP subtype-selective agonists and antagonists suggested EP4 mediates the prostaglandin-induced tube formation, and this conclusion was substantiated with small interfering RNA to specifically knockdown the EP4 expression. EP4 couples to Gαs, leading to activation of protein kinase A (PKA). Inhibition of PKA activity or knockdown of PKA catalytic subunit γ with RNAi attenuates the PGE2-induced tube formation. Further, knocking down the expression of Rap1A, HSPB6, or endothelial NO synthase, which serve as PKA-activatable substrates, inhibits the tube formation, whereas knockdown of RhoA or glycogen synthase kinase 3β that are inactivated after phosphorylation by PKA increases the tube formation. These results support the existence of EP4-to-PKA angiogenic signal and provide rationale for use of selective EP4 signal inhibitors as a probable strategy to control pathologic angiogenesis.     

Macrophage migration inhibitory factor upregulates angiogenic factors and correlates with clinical measures in rheumatoid arthritis

OBJECTIVE: To investigate the relationship between macrophage migration inhibitory factor (MIF) levels and clinical measures in rheumatoid arthritis (RA), and the potential for regulation of angiogenesis in RA. METHODS: Serum and synovial fluid (SF) levels of MIF and vascular endothelial growth factor (VEGF) in patients with RA were determined by sandwich ELISA, and the relationships among MIF, VEGF, and RA clinical measures were analyzed. RA synovial fibroblasts were cultured with recombinant human MIF (rhMIF) and the production of VEGF and interleukin 8 (IL-8) were measured in the conditioned media. The angiogenic effect of MIF was examined using established measures of angiogenesis in vitro. RESULTS: Erythrocyte sedimentation rate, C-reactive protein, and the daily dosage of oral prednisolone were correlated with SF levels of MIF. The SF levels of MIF were found to be higher in patients with bony erosion than in those without (69.2 +/- 11.4 ng/ml vs 44.0 +/- 6.2 ng/ml; p = 0.045). MIF levels had good correlation with VEGF levels (r = 0.52, p < 0.001 in sera, and r = 0.6, p < 0.001 in SF). Production of the angiogenic factors VEGF and IL-8 was enhanced in cultured RA synovial fibroblasts stimulated by rhMIF. Endothelial tube formation was augmented when the endothelial cells were cultured with the conditioned media from rhMIF-pretreated SF mononuclear cells, and this phenomenon was reversed by anti-VEGF antibody. CONCLUSION: SF MIF may reflect the clinical activity in patients with RA, and rhMIF induces the angiogenic factors in RA synovial fibroblasts. These results suggest that MIF may be an important cytokine in the perpetuation of the angiogenic and inflammatory processes in patients with RA.