Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38+/-6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 microM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration.     

Lysophosphatidylglycerol stimulates chemotactic migration and tube formation in human umbilical vein endothelial cells

In this study, we observed that lysophosphatidylglycerol (LPG) stimulates extracellular signal-regulated kinase (ERK) in human umbilical vein endothelial cells (HUVECs). LPG-stimulated ERK activity was not inhibited by pertussis toxin (PTX), indicating PTX-sensitive G-proteins-independent manner. In terms of functional aspect, LPG induced chemotactic migration of HUVECs in a PTX-insensitive manner. Preincubation of HUVECs with an ERK inhibitor (PD98059) completely inhibited LPG-induced chemotactic migration, suggesting the crucial role of ERK in the process. LPG-induced ERK activation and chemotactic migration in HUVECs were not affected by an lysophosphatidic acid receptor-selective antagonist (Ki16425), indicating lysophosphatidic acid receptors-independency. We also found that LPG stimulated tube formation in HUVECs. Taken together we suggest that LPG stimulates HUVECs and result in chemotactic migration and tube formation, suggesting a new aspect of LPG as a modulator of endothelial cell functioning.     

The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells

Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted the proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs.     

Saikosaponin C induces endothelial cells growth, migration and capillary tube formation

Saikosaponin C is one of the saikosaponins that are consisted in a Chinese herb, Radix Bupleuri. Recently, saikosaponins have been reported to have properties of cell growth inhibition, inducing cancer cells differentiation and apoptosis. However, saikosaponin C had no correlation with cell growth inhibition. In this study, we investigated the role of saikosaponin C on the growth of endothelial cells and angiogenesis. We found that saikosaponin C yielded a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. In addition to inducing endothelial cells growth, saikosaponin C also induced endothelial cells migration and capillary tube formation. The gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and the p42/p44 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells growth, migration and angiogenesis were also induced by saikosaponin C. From these results, we suggest that saikosaponin C may have the potential for therapeutic angiogenesis but is not suitable for cancer therapy.     

Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.     

Differential effects of Heparitinase I and Heparitinase III on endothelial tube formation in vitro

Heparan sulfate proteoglycans (HSPGs) play vital roles in many steps of angiogenesis under physiological and pathological conditions. HSPGs on endothelial cell surfaces act as co-receptors for a variety of pro-angiogenic growth factors such as FGF and VEGF and anti-angiogenic factors such as endostatin. However, the fine structural requirements of these binding interactions are dependent on the sulfation patterns of HSPGs. Previous studies have shown that Heparitinases, heparin lyases isolated from Flavobacterium heparinum, can cleave heparan sulfate chains. These enzymes have been shown to reduce tumor—derived neovascularization in vivo in mice. However, the results from these experiments could not conclusively pinpoint the origin of the HS fragments. Thus, in this study we utilized an in vitro assay to assess the differential effects of Heparitinase I (Hep I) and Heparitinase III (Hep III) on endothelial tube formation. Hep III was found to be a more potent inhibitor of tube formation than Hep I. In conclusion, differential cleavage of endothelial cell surface bound HS can affect the extent of inhibition of tube formation.     

PPAR activators inhibit endothelial cell migration by targeting Akt

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose metabolism and exert several vascular effects that may provide a dual benefit of these receptors on metabolic disorders and atherosclerotic vascular disease. Endothelial cell migration is a key event in the pathogenesis of atherosclerosis. We therefore investigated the effects of lipid-lowering PPARalpha-activators (fenofibrate, WY14643) and antidiabetic PPARgamma-activators (troglitazone, ciglitazone) on this endothelial cell function. Both PPARalpha- and PPARgamma-activators significantly inhibited VEGF-induced migration of human umbilical vein endothelial cells (EC) in a concentration-dependent manner. Chemotactic signaling in EC is known to require activation of two signaling pathways: the phosphatidylinositol-3-kinase (PI3K)-->Akt- and the ERK1/2 mitogen-activated protein kinase (ERK MAPK) pathway. Using the pharmacological PI3K-inhibitor wortmannin and the ERK MAPK-pathway inhibitor PD98059, we observed a complete inhibition of VEGF-induced EC migration. VEGF-induced Akt phosphorylation was significantly inhibited by both PPARalpha- and gamma-activators. In contrast, VEGF-stimulated ERK MAPK-activation was not affected by any of the PPAR-activators, indicating that they inhibit migration either downstream of ERK MAPK or independent from this pathway. These results provide first evidence for the antimigratory effects of PPAR-activators in EC. By inhibiting EC migration PPAR-activators may protect the vasculature from pathological alterations associated with metabolic disorders.     

Angiotensin II-induced smooth muscle cell migration is mediated by LDL receptor-related protein 1 via regulation of matrix metalloproteinase 2 expression

Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.     

Homocysteine modulates the proteolytic potential of human vascular endothelial cells

Pathological levels of homocysteine induce a metalloproteinase-dependent degradation of the elastic structures in arterial wall. This elastolytic process is preferentially localized toward the internal elastic laminae and in the first layers of the media, suggesting endothelium could participate in extracellular matrix degradation induced by homocysteine. Therefore, we studied the effects of homocysteine on proteolytic potential of endothelial cells. Human umbilical vein endothelial cells were cultured with concentrations of homocysteine matching human physiological (10 microM) and pathological (50, 100, and 250 microM) plasma homocysteine levels. Pathological levels of homocysteine increased the secretion of elastolytic metalloproteinase-2 and -9, but not of metalloproteinase-3 and -7. Homocysteine also increased the expression of human tissue kallikrein, a potential activator of matrix metalloproteinase-2 and -9, while the expression of urokinase plasminogen activator was not altered. These results suggest vascular endothelial cells could participate in the subendothelial degradation of the arterial elastic structures occurring in hyperhomocysteinemia.     

RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.     

Low-dose aspirin promotes endothelial progenitor cell migration and adhesion and prevents senescence

Circulating endothelial progenitor cells (EPCs) play a key role in restoring endothelial function and enhancing angiogenesis. However, the effects of low-dose aspirin on circulating EPCs are not well known. We investigated the effects of low-dose aspirin on EPC migration, adhesion, senescence, proliferation, apoptosis and endothelial nitric oxide synthase (eNOS) expression. EPC migration was detected by a modified Boyden chamber assay. EPC adhesion assay was performed by counting adherent cells on fibronectin-coated culture dishes. EPC senescence was assessed by both senescence-associated-beta-galactosidase staining and DAPI staining. EPC proliferation was analyzed by MTT assay. EPC apoptosis was evaluated by flow cytometric analysis. eNOS protein expression was measured by Western blotting analysis. Aspirin promoted EPC migratory and adhesive capacity at concentrations between 0.1 and 100micromol/L and prevented senescence at concentrations between 50 and 100micromol/L. Meanwhile, aspirin in a range of these concentrations did not affect EPC proliferation, apoptosis or eNOS expression. Our findings indicate that low-dose aspirin promotes migration and adhesion and delays the onset of senescence of EPCs.     

Angiopoietin-2 stimulates migration of endothelial progenitors and their interaction with endothelium

The factors controlling recruitment of endogenous and transplanted endothelial progenitor cells (EPC) to areas of neovascularization are largely unknown. In this study, we have examined the possibility that EPC migration and adhesion could be regulated by angiopoietin-2 (Ang2), a soluble ligand expressed by endothelial cells at sites of vessel remodelling and angiogenesis. We show for the first time that Ang2 causes a marked stimulation of EPC migration. This was specific for EPC as the ligand failed to affect endothelial cell migration. Ang2-stimulated EPC migration was inhibited by soluble Tie2 ectodomain. Furthermore, the ligand stimulated adhesion between EPC and endothelial monolayers.     

Melanoma cell-derived vascular endothelial growth factor induces endothelial tubulogenesis within fibrin gels by a metalloproteinase-mediated mechanism

Angiogenesis is a process required not only for embryonal development but is encountered in wound healing and in pathological situations such as tumour growth. In vitro, formation of capillary-like structures can be induced by seeding human microvascular endothelial cells (HDMECs) on top of a fibrin matrix in the presence of phorbol 12-myristate 13-acetate (PMA) as a stimulating agent. In this study, we show that supernatants collected from high-invasive melanoma cells (BLM) induce the formation of tubular structures similar to PMA treatment whereas supernatants from low-invasive cells (WM164) did not. Analysis of proteins secreted into the supernatant of both melanoma cell lines identified differential expression of several pro-angiogenic proteins in high- and low-invasive melanoma cells. Vascular endothelial growth factor (VEGF) was strongly expressed by high- but not by low-invasive melanoma cells. Neutralisation of VEGF as well as inhibition of matrix metalloproteases (MMPs) using the broad spectrum MMP inhibitor 1,10-phenanthroline, both strongly reduced the melanoma-induced tube formation. PMA treatment of HDMECs on a fibrin matrix stimulated MT1-MMP synthesis, indicating that this protease is involved in PMA-induced angiogenesis. In addition, stimulation of HDMECs by supernatants of BLM melanoma cells resulted in a strong induction of ADAM-15, which is known to act as a metalloproteinase. In conclusion, these results show that VEGF released by melanoma cells is an important mediator of neo-vascularisation and that this process depends on the presence of metalloproteinases.     

Methylmercury-injury effect on tube formation by cultured human vascular endothelial cells

The effect of methylmercury chloride (MeHg) on growth and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were collected by enzymatic digestion with collagenase. Precultivation of HUVECs with MeHg at concentrations of 1.0–50.0 mol/L exerted negligible effects on the viable cell number, while the viable cell number was slightly reduced at 100 mol/L and fell to zero at concentrations exceeding 500.0 mol/L MeHg. The viable cell number was depressed in a concentration-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 mol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal bovine serum (FBS) increased tube formation in a dose-dependent manner, with half-maximum stimulation of tube formation at approximately 3.4% FBS. The length of tube formation decreased time-dependently at concentrations of 0.1 and 1.0 mol/L MeHg. Pretreatment of Matrigel with 1 mol/L MeHg before the cell seeding reduced the tube formation by HUVECs. These results suggest that the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function.Abbreviations MeHg methylmercury chloride - HUVECs human umbilical vein endothelial cells     

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A₁₆₅). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.     

PRKX critically regulates endothelial cell proliferation, migration, and vascular-like structure formation

Angiogenesis is a fundamental step in several important physiological events and pathological conditions including embryonic development, wound repair, tumor growth and metastasis. PRKX was identified as a novel type-I cAMP-dependent protein kinase gene expressed in multiple developing tissues. PRKX has also been shown to be phylogenetically and functionally distinct from PKA. This study presents the first evidence that PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation, which are the three essential processes for angiogenesis. In contrast, classic PKA demonstrated an inhibitory effect on endothelia vascular-like structure formation. Our findings suggest that PRKX is an important protein kinase engaged in the regulation of angiogenesis and could play critical roles in various physiological and pathological conditions involving angiogenesis. PRKX binds to Pin-1, Magi-1 and Bag-3, which regulate cell proliferation, apoptosis, differentiation and tumorigenesis. The interaction of PRKX with Pin-1, Magi-1 and Bag-3 could contribute to the stimulating role of PRKX in angiogenesis.