Atorvastatin induces tissue transglutaminase in human endothelial cells

Tissue transglutaminase (tTgase) contributes to the organisation of the basement membrane and is therefore thought to be important for the integrity and stability of the vessel wall. In the present study, we hypothesised that the HMG-CoA reductase inhibitor atorvastatin may up-regulate the tTgase expression in endothelial cells and thereby exert beneficial effects on endothelial function. Treatment of human umbilical vein endothelial cells (HUVEC) with atorvastatin (1-10 microM) caused a clear increased expression of tTgase in both permeabilised and non-permeabilised HUVEC. In contrast, stimulation of HUVEC with TNFalpha had no substantial effect on tTgase expression or localisation but inhibited the atorvastatin-induced up-regulation and externalisation of tTgase. Propidium iodide staining revealed that statin-induced apoptosis is not responsible for the enhanced expression. By inducing the expression of tTgase, statins may promote tTgase-mediated stabilisation of the basement membrane. This effect of atorvastatin may contribute to the beneficial role of statins on endothelial function.     

Homocysteine inhibits the proliferation and invasive potential of HT-1080 human fibrosarcoma cells

The impairment of homocysteine metabolism has been related to several disorders and diseases. Recently, homocysteine has been shown to inhibit key steps of angiogenesis, including endothelial cell proliferation, invasion, and remodeling of the extracellular matrix. Since these are also key steps in tumor invasion and metastasis, it can be hypothesized that homocysteine can also interfere in these processes. Therefore, we studied the effects of homocysteine on tumor proliferation and invasion, as well as on urokinase, a key extracellular matrix-degrading protease, using a model human tumor cell line. This study demonstrates that, in fact, homocysteine inhibits HT-1080 proliferation and invasion, and is a potent inhibitor of tumor cell urokinase expression.     

Anti-angiogenic effects of homocysteine on cultured endothelial cells

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.     

Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.     

Homocysteine transport by human aortic endothelial cells: identification and properties of import systems

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Transport of L-homocysteine into and out of the human vascular endothelium is poorly understood. We hypothesized that cultured human aortic endothelial cells (HAEC) would import L-homocysteine on one or more of the L-cysteine transport systems. Inhibitors of the transporters were used to characterize the uptake of [35S]L-homocysteine, [35S]L-homocystine, and [35S]L-cysteine. We found that L-homocysteine uptake is mediated by the sodium-dependent cysteine transport systems X(AG), ASC, and A, and the sodium-independent transport system L. Thus, HAEC utilize multiple cysteine transporters (X(AG) > or = L > ASC > A) to import L-homocysteine. Kinetic analysis supported the uptake results. Michaelis-Menten constants (Km) for the four systems yielded values of 19.0, 27.1, 112, and 1000 microM for systems L, X(AG), ASC, and A, respectively. The binding and uptake of [35S]L-homocystine, the disulfide homodimer of L-homocysteine, was mediated by systems X(AG), L, and ASC but not by system A. In contrast to [35S]L-homocysteine, system x(c) was active for [35S]L-homocystine uptake. A similar pattern was observed for [35S]L-cysteine. Thus, L-homocysteine and L-homocystine found in hyperhomocysteinemic subjects can gain entry into the vascular endothelium by way of multiple L-cysteine transporters.     

Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.     

Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.     

Cyclic strain-mediated regulation of vascular endothelial cell migration and tube formation

Hemodynamic forces exerted by blood flow (cyclic strain, shear stress) affect the initiation and progression of angiogenesis; however, the precise signaling mechanism(s) involved are unknown. In this study, we examine the role of cyclic strain in regulating bovine aortic endothelial cell (BAEC) migration and tube formation, indices of angiogenesis. Considering their well-documented mechanosensitivity, functional inter-dependence, and involvement in angiogenesis, we hypothesized roles for matrix metalloproteinases (MMP-2/9), RGD-dependent integrins, and urokinase plasminogen activator (uPA) in this process. BAECs were exposed to equibiaxial cyclic strain (5% strain, 1Hz for 24h) before their migration and tube formation was assessed by transwell migration and collagen gel tube formation assays, respectively. In response to strain, both migration and tube formation were increased by 1.83+/-0.1- and 1.84+/-0.1-fold, respectively. Pertussis toxin, a Gi-protein inhibitor, decreased strain-induced migration by 45.7+/-32% and tube formation by 69.8+/-13%, whilst protein tyrosine kinase (PTK) inhibition with genistein had no effect. siRNA-directed attenuation of endothelial MMP-9 (but not MMP-2) expression/activity decreased strain-induced migration and tube formation by 98.6+/-41% and 40.7+/-31%, respectively. Finally, integrin blockade with cRGD peptide and siRNA-directed attenuation of uPA expression reduced strain-induced tube formation by 85.7+/-15% and 84.7+/-31%, respectively, whilst having no effect on migration. CONCLUSIONS: Cyclic strain promotes BAEC migration and tube formation in a Gi-protein-dependent PTK-independent manner. Moreover, we demonstrate for the first time a putative role for MMP-9 in both strain-induced events, whilst RGD-dependent integrins and uPA appear only to be involved in strain-induced tube formation.     

Homocysteine in microvascular endothelial cell barrier permeability

Redox stress activates the endothelium and upregulates matrix metalloproteinases (MMPs), which degrade the matrix and lead to blood-endothelial barrier leakage. Interestingly, elevated levels of plasma homocysteine (Hcy) are associated with vascular dementia, seizure, stroke, and Alzheimer disease. Hcy competes with the γ-aminobutyric acid (GABA)-A/B receptors and behave like an excitatory neurotransmitter. GABA stimulates the inhibitory neurotransmitter GABA-A/B receptor and decreases arterial blood pressure. However, the neural mechanisms of microvascular remodeling in hyperhomocysteinemia are unclear. This review addresses the idea that Hcy induces microvascular permeability by attenuating the GABA-A/B receptors and increasing redox stress, which activates a disintegrin and metalloproteinase that suppresses tissue inhibitors of metalloproteinase. This process causes disruption of the matrix in the blood-brain barrier. Understanding the mechanism of Hcy-mediated changes in permeability of the blood-brain barrier and extracellular matrix that can alter the neuronal environment in cerebral-vascular dementia is of great importance in developing treatments for this disease.     

Differentiation of human dermal fibroblasts towards endothelial cells

The ultimate goal of vascular tissue engineering is the production of functional grafts for clinical use. Difficulties acquiring autologous endothelial cells have motivated the search for alternative cell sources. Differentiation of dermal fibroblasts towards several mesenchymal lineages as well as endothelial cells has been proposed. The aim of the present study was to investigate the endothelial differentiation capacity of human dermal fibroblasts on a gene expression, protein expression and functional physiological level. Endothelial differentiation of fibroblasts was induced by culturing cells in 30% human serum, but not in fetal calf serum. Expression of proteins and genes relevant for endothelial function and differentiation was increased after induction. Furthermore, fibroblasts exposed to 30% human serum displayed increased uptake of low-density lipoprotein and formation of capillary-like networks. The results of this study may have an impact on cell sourcing for vascular tissue engineering, and the development of methods for vascularization of autologous tissue engineered constructs.     

UVA-mediated down-regulation of MMP-2 and MT1-MMP coincides with impaired angiogenic phenotype of human dermal endothelial cells

UVA irradiation, dose-dependently (5-20 J/cm2), was shown to impair the morphogenic differentiation of human microvascular endothelial cells (HMECs) on Matrigel. Parallely, UVA down-regulated the expression of MMP-2 and MT1-MMP, both at the protein and the mRNA levels. On the contrary, the production of MMP-1 and TIMP-1 by HMECs increased following UVA treatment. The inhibitory effect of UVA on MMP expression and pseudotubes formation was mediated by UVA-generated singlet oxygen (1O2). The contribution of MT1-MMP, but not TIMP-1, to the regulation of HMECs' angiogenic phenotype following UVA irradiation was suggested using elastin-derived peptides and TIMP-1 blocking antibody, respectively.     

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor has various cellular effects such as cell growth and apoptosis. In the present study, we evaluated the effects of SBHA on the growth and death of A549 lung cancer cells. SBHA inhibited the growth of A549 cells with an IC₅₀ of approximately 50 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that SBHA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, as evidenced by sub-G1 cells and annexin V-FITC staining cells. SBHA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, Bax increase, and the activation of caspase-3. All of the tested caspase inhibitors significantly rescued some cells from SBHA-induced A549 cell death. However, none of the caspase inhibitors prevented the loss of MMP (ΔΨ_m) induced by SBHA. Intracellular reactive oxygen species (ROS) levels including O ₂ ^?? were increased in 50 μM SBHA-treated A549 cells. None of the caspase inhibitors attenuated ROS levels in these cells. SBHA also elevated the number of glutathione (GSH)-depleted cells in A549 cells, which was reduced by treatment with caspase inhibitors. In conclusion, this is the first report that SBHA inhibited the growth of A549 lung cancer cells via caspase-dependent apoptosis, which was related to GSH depletion rather than changes in ROS level.     

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.     

Homocysteine pre-treatment increases redox capacity in both endothelial and tumor cells

Objective: We studied the modulatory effects of homocysteine pre-treatment on the disulfide reduction capacity of tumor and endothelial cells.

Methods: Human MDA-MB-231 breast carcinoma and bovine aorta endothelial cells were pre-treated for 1–24 hours with 0.5–5 mM homocysteine or homocysteine thiolactone. After washing to eliminate any rest of homocysteine or homocysteine thiolactone, cell redox capacity was determined by using a method for measuring disulfide reduction.

Results: Homocysteine pre-treatments for 1–4 hours at a concentration of 0.5–5 mM increase the disulfide reduction capacity of both tumor and endothelial cells. This effect cannot be fully mimicked by either cysteine or homocysteine thiolactone pre-treatments of tumor cells.

Discussion: Taken together, our data suggest that homocysteine can behave as an anti-oxidant agent by increasing the anti-oxidant capacity of tumor and endothelial cells.     

Induction of apoptotic cell death in vascular endothelial cells cultured in three-dimensional collagen lattice

Endothelial cells derived from fetal bovine aorta (BAECs) undergo apoptosis in three-dimensional (3-D) type I collagen lattice in the absence of specific angiogenic factor. In the presence of angiogenic factor, BAECs survive and form a capillary-like tube structure in 3-D culture. In the present study we elucidate the mechanisms of BAECs apoptosis or survival and tube formation in 3-D culture. When BAECs embedded in collagen lattice were cultured with angiogenic factor (fibroblast growth factor-2 (FGF-2) or 4beta-phorbol 12-myristate 13-acetate (PMA)) in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, BAECs did not form tube structures and underwent apoptosis in collagen lattice. Function-blocking antibody against alphavbeta3 integrin also inhibited tube formation and induced apoptosis in 3-D culture in the presence of angiogenic factors. Exposure of BAECs to FGF-2 and PMA had no effect on the alphavbeta3 integrin expression but induced the activation of alphavbeta3 integrin. PD98059 attenuated alphavbeta3 integrin activation in response to angiogenic factor. KB-R8301, a hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, prevented apoptotic cell death in the absence of angiogenic factor in 3-D culture and enhanced capillary-like tube formation in the presence of angiogenic factor, which was not inhibited by the anti-alphavbeta3 integrin antibody. The results suggest that angiogenic factor-induced alphavbeta3 integrin activation through the MEK-ERK pathway regulates the BAEC fate between apoptosis and angiogenesis in collagen lattice. MMP derived from BAECs seems to play a key role in the release of cryptic ligands for alphavbeta3 integrin from intact collagen.     

Modulation of angiogenesis by tissue inhibitor of metalloproteinase-4

Despite the importance of MMP activity in the regulation of angiogenesis, relatively little is known about the role of TIMP-4, the most recently discovered endogenous MMP inhibitor, in modulating neovascularization. It has largely been assumed that all TIMPs are capable of inhibiting angiogenesis in vivo. However, it is now widely appreciated that TIMPs-1, -2, and -3 differ significantly in their ability to modulate angiogenic processes in vitro and angiogenesis in vivo. In order to study the effect of TIMP-4 in controlling angiogenesis, we have cloned and expressed TIMP-4 in a Pichia pastoris expression system, purified it to homogeneity, and tested its ability to regulate angiogenesis in vivo and in vitro. Our studies demonstrate that TIMP-4 is an inhibitor of capillary endothelial cell migration, but not of proliferation or of angiogenesis in vivo.     

Silencing of calpain expression reduces the metastatic potential of human osteosarcoma cells

Osteosarcoma, the most common primary bone tumor in young adults, is characterized by local invasion and distant metastasis. But detailed mechanisms of tumorigenicity and metastasis of osteosarcoma are not well known. We report the involvement of calpains, a family of calcium-activated, cysteine proteases, in the invasive and metastatic processes of human osteosarcoma cells. By using siRNA treatment, the expression of μ- and m-calpains were downregulated in human Saos-2 osteosarcoma cells. Both the adhesive and invasive potentials were significantly attenuated in calpain siRNA-transfected human Saos-2 osteosarcoma cells. MMPs are the main factors involved in malignant tumor invasion and metastasis. siRNA of calpains also significantly inhibited the secretion of MMP-2 in Saos-2 cells. These results suggest that μ- and m-calpains are important in the invasion and metastasis of human osteosarcoma cells, and calpains might be targeted to reduce tumor progression.     

Immunohistochemical localization of endothelin in human vascular endothelial cells

The cellular localization of endothelin (ET), a novel vasoconstrictor peptide, was studied in human vascular tissues by immunohistochemistry. Distinct and diffuse staining for ET-like immunoreactivity was demonstrated in the cytoplasm of vascular endothelial cells, but not in smooth muscle cells or adventitial fibroblasts. The specificity was confirmed by the negative results following immunoabsorption. These findings suggest that human vascular endothelial cells function as an endocrine and/or paracrine cells for ET secretion.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

Characterization of matrix metalloproteinase expressed by human embryonic kidney cells

There is little information available on the proteases expressed by human embryonic kidney (HEK) cells, which are often used for expression of recombinant proteins and production of adenovirus vector. The expression profile of proteases in HEK cell line was investigated using zymography, mRNA analysis, western blotting and protein array. The major protease was gelatinase A [or matrix metalloproteinase (MMP)-2]. Beside, other MMPs, such as MMP-1, -2, -3, -8, -9, -10, -13 and membrane type (MT) 1- and 3−MMP, as well as tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, were also expressed by HEK cells. Characterization of MMP and TIMP profiles expressed by HEK cells provides the basis for degradation control of recombinant protein and adenovirus vector during culture and purification processes.