Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe ^−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe ^−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.     

Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

Background

Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H₂O₂) in postnatal neovascularization.

Methodology/Principal Findings

We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H₂O₂ in ECs in post-ischemic neovascularization in vivo. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H₂O₂ in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.

Conclusions/Significance

Endogenous ECs-derived H₂O₂ plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.     

Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model

Background

Cilostazol(CLZ) has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM)-derived endothelial progenitor cell (EPC) contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs.

Methodology/Principal Findings

Balloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury.

Conclusions/Significance

CLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for endothelial regeneration, which is a key event for preventing atherosclerosis or restenosis after vascular intervention.     

Endothelial cell-specific molecule 2 (ECSM2) localizes to cell-cell junctions and modulates bFGF-directed cell migration via the ERK-FAK pathway

Background

Despite its first discovery by in silico cloning of novel endothelial cell-specific genes a decade ago, the biological functions of endothelial cell-specific molecule 2 (ECSM2) have only recently begun to be understood. Limited data suggest its involvement in cell migration and apoptosis. However, the underlying signaling mechanisms and novel functions of ECSM2 remain to be explored.

Methodology/Principal Findings

A rabbit anti-ECSM2 monoclonal antibody (RabMAb) was generated and used to characterize the endogenous ECSM2 protein. Immunoblotting, immunoprecipitation, deglycosylation, immunostaining and confocal microscopy validated that endogenous ECSM2 is a plasma membrane glycoprotein preferentially expressed in vascular endothelial cells (ECs). Expression patterns of heterologously expressed and endogenous ECSM2 identified that ECSM2 was particularly concentrated at cell-cell contacts. Cell aggregation and transwell assays showed that ECSM2 promoted cell-cell adhesion and attenuated basic fibroblast growth factor (bFGF)-driven EC migration. Gain or loss of function assays by overexpression or knockdown of ECSM2 in ECs demonstrated that ECSM2 modulated bFGF-directed EC motility via the FGF receptor (FGFR)-extracellular regulated kinase (ERK)-focal adhesion kinase (FAK) pathway. The counterbalance between FAK tyrosine phosphorylation (activation) and ERK-dependent serine phosphorylation of FAK was critically involved. A model of how ECSM2 signals to impact bFGF/FGFR-driven EC migration was proposed.

Conclusions/Significance

ECSM2 is likely a novel EC junctional protein. It can promote cell-cell adhesion and inhibit bFGF-mediated cell migration. Mechanistically, ECSM2 attenuates EC motility through the FGFR-ERK-FAK pathway. The findings suggest that ECSM2 could be a key player in coordinating receptor tyrosine kinase (RTK)-, integrin-, and EC junctional component-mediated signaling and may have important implications in disorders related to endothelial dysfunction and impaired EC junction signaling.     

Phloroglucinol inhibits the bioactivities of endothelial progenitor cells and suppresses tumor angiogenesis in LLC-tumor-bearing mice

Background

There is increasing evidence that phloroglucinol, a compound from Ecklonia cava, induces the apoptosis of cancer cells, eventually suppressing tumor angiogenesis.

Methodology/Principal Findings

This is the first report on phloroglucinol''s ability to potentially inhibit the functional bioactivities of endothelial progenitor cells (EPCs) and thereby attenuate tumor growth and angiogenesis in the Lewis lung carcinoma (LLC)-tumor-bearing mouse model. Although Phloroglucinol did not affect their cell toxicity, it specifically inhibited vascular endothelial growth factor (VEGF) dependent migration and capillary-like tube formation of EPCs. Our matrigel plug assay clearly indicated that orally injected phloroglucinol effectively disrupts VEGF-induced neovessel formation. Moreover, we demonstrated that when phloroglucinol is orally administered, it significantly inhibits tumor growth and angiogenesis as well as CD45⁻/CD34⁺ progenitor mobilization into peripheral blood in vivo in the LLC-tumor-bearing mouse model.

Conclusions/Significance

These results suggest a novel role for phloroglucinol: Phloroglucinol might be a modulator of circulating EPC bioactivities, eventually suppressing tumorigenesis. Therefore, phloroglucinol might be a candidate compound for biosafe drugs that target tumor angiogenesis.     

Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis

Background

Endothelial Progenitor Cells (EPC) support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors.

Objective

The aim of this study was to investigate the role of PDGF-BB and PDGFRβ in EPC-mediated angiogenesis of differentiated endothelial cells.

Methods and Results

Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01). EPC-CM increased proliferation (1.39-fold; P<0.001) and migration (2.13-fold; P<0.001) of isolated human umbilical vein endothelial cells (HUVEC), as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01). The capacity of EPC-CM to modulate the PDGFRβ expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01). EPC-CM triggered a distinct up-regulation of PDGFRβ (2.5±0.5; P<0.05) and its phosphorylation (3.6±0.6; P<0.05) in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone.

Conclusion

These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGF/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.     

Zinc-chelation contributes to the anti-angiogenic effect of ellagic acid on inhibiting MMP-2 activity, cell migration and tube formation

Background

Ellagic acid (EA), a dietary polyphenolic compound, has been demonstrated to exert anti-angiogenic effect but the detailed mechanism is not yet fully understood. The aim of this study was to investigate whether the zinc chelating activity of EA contributed to its anti-angiogenic effect.

Methods and Principal Findings

The matrix metalloproteinases-2 (MMP-2) activity, a zinc-required reaction, was directly inhibited by EA as examined by gelatin zymography, which was reversed dose-dependently by adding zinc chloride. In addition, EA was demonstrated to inhibit the secretion of MMP-2 from human umbilical vein endothelial cells (HUVECs) as analyzed by Western blot method, which was also reversed by the addition of zinc chloride. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), known to down-regulate the MMP-2 activity, was induced by EA at both the mRNA and protein levels which was correlated well with the inhibition of MMP-2 activity. Interestingly, zinc chloride could also abolish the increase of EA-induced RECK expression. The anti-angiogenic effect of EA was further confirmed to inhibit matrix-induced tube formation of endothelial cells. The migration of endothelial cells as analyzed by transwell filter assay was suppressed markedly by EA dose-dependently as well. Zinc chloride could reverse these two effects of EA also in a dose-dependent manner. Since magnesium chloride or calcium chloride could not reverse the inhibitory effect of EA, zinc was found to be involved in tube formation and migration of vascular endothelial cells.

Conclusions/Significance

Together these results demonstrated that the zinc chelation of EA is involved in its anti-angiogenic effects by inhibiting MMP-2 activity, tube formation and cell migration of vascular endothelial cells. The role of zinc was confirmed to be important in the process of angiogenesis.     

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells

Background

Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.

Methodology

We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.

Results and Conclusion

Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8–12 hr of continuous shear-cytokine activation contrary to the commonly observed 4–6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.     

Up-regulation of NDRG2 in senescent lens epithelial cells contributes to age-related cataract in human

Background

Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer''s disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract.

Methodology/Principal Findings

Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H₂O₂ to simulate senescence. After being exposed to 50 µM H₂O₂ for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50–200 µM H₂O₂ for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses.

Conclusions/Significance

NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.     

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background

One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape.

Methodology/Principal Findings

We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally.

Conclusions/Significance

The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development.     

High resolution imaging of vascular function in zebrafish

Rationale

The role of the endothelium in the pathogenesis of cardiovascular disease is an emerging field of study, necessitating the development of appropriate model systems and methodologies to investigate the multifaceted nature of endothelial dysfunction including disturbed barrier function and impaired vascular reactivity.

Objective

We aimed to develop and test an optimized high-speed imaging platform to obtain quantitative real-time measures of blood flow, vessel diameter and endothelial barrier function in order to assess vascular function in live vertebrate models.

Methods and Results

We used a combination of cutting-edge optical imaging techniques, including high-speed, camera-based imaging (up to 1000 frames/second), and 3D confocal methods to collect real time metrics of vascular performance and assess the dynamic response to the thromboxane A₂ (TXA₂) analogue, U-46619 (1 µM), in transgenic zebrafish larvae. Data obtained in 3 and 5 day post-fertilization larvae show that these methods are capable of imaging blood flow in a large (1 mm) segment of the vessel of interest over many cardiac cycles, with sufficient speed and sensitivity such that the trajectories of individual erythrocytes can be resolved in real time. Further, we are able to map changes in the three dimensional sizes of vessels and assess barrier function by visualizing the continuity of the endothelial layer combined with measurements of extravasation of fluorescent microspheres.

Conclusions

We propose that this system-based microscopic approach can be used to combine measures of physiologic function with molecular behavior in zebrafish models of human vascular disease.     

Diosgenin, a steroidal saponin, inhibits migration and invasion of human prostate cancer PC-3 cells by reducing matrix metalloproteinases expression

Background

Diosgenin, a steroidal saponin obtained from fenugreek (Trigonella foenum graecum), was found to exert anti-carcinogenic properties, such as inhibiting proliferation and inducing apoptosis in a variety of tumor cells. However, the effect of diosgenin on cancer metastasis remains unclear. The aim of the study is to examine the effect of diosgenin on migration and invasion in human prostate cancer PC-3 cells.

Methods and Principal Findings

Diosgenin inhibited proliferation of PC-3 cells in a dose-dependent manner. When treated with non-toxic doses of diosgenin, cell migration and invasion were markedly suppressed by in vitro wound healing assay and Boyden chamber invasion assay, respectively. Furthermore, diosgenin reduced the activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatin zymography assay. The mRNA level of MMP-2, -9, -7 and extracellular inducer of matrix metalloproteinase (EMMPRIN) were also suppressed while tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased by diosgenin. In addition, diosgenin abolished the expression of vascular endothelial growth factor (VEGF) in PC-3 cells and tube formation of endothelial cells. Our immunoblotting assays indicated that diosgenin potently suppressed the phosphorylation of phosphatidylinositide-3 kinase (PI3K), Akt, extracellular signal regulating kinase (ERK) and c-Jun N-terminal kinase (JNK). In addition, diosgenin significantly decreased the nuclear level of nuclear factor kappa B (NF-κB), suggesting that diosgenin inhibited NF-κB activity.

Conclusion/Significance

The results suggested that diosgenin inhibited migration and invasion of PC-3 cells by reducing MMPs expression. It also inhibited ERK, JNK and PI3K/Akt signaling pathways as well as NF-κB activity. These findings reveal new therapeutic potential for diosgenin in anti-metastatic therapy.     

Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis

Background

Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.

Methodology/Principal Findings

In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.

Conclusions/Significance

These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.     

The antiangiogenic 16K prolactin impairs functional tumor neovascularization by inhibiting vessel maturation

Background

Angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology.

Methodology/Principal Findings

Here we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling.

Conclusions/Significance

Taken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.     

Nanoporous CREG-Eluting Stent Attenuates In-Stent Neointimal Formation in Porcine Coronary Arteries

Background

The goal of this study was to evaluate the efficacy of a nanoporous CREG-eluting stent (CREGES) in inhibiting neointimal formation in a porcine coronary model.

Methods

In vitro proliferation assays were performed using isolated human endothelial and smooth muscle cells to investigate the cell-specific pharmacokinetic effects of CREG and sirolimus. We implanted CREGES, control sirolimus-eluting stents (SES) or bare metal stents (BMS) into pig coronary arteries. Histology and immunohistochemistry were performed to assess the efficacy of CREGES in inhibiting neointimal formation.

Results

CREG and sirolimus inhibited in vitro vascular smooth muscle cell proliferation to a similar degree. Interestingly, human endothelial cell proliferation was only significantly inhibited by sirolimus and was increased by CREG. CREGES attenuated neointimal formation after 4 weeks in porcine coronary model compared with BMS. No differences were found in the injury and inflammation scores among the groups. Scanning electron microscopy and CD31 staining by immunohistochemistry demonstrated an accelerated reendothelialization in the CREGES group compared with the SES or BMS control groups.

Conclusions

The current study suggests that CREGES reduces neointimal formation, promotes reendothelialization in porcine coronary stent model.     

Globotriaosylsphingosine accumulation and not alpha-galactosidase-A deficiency causes endothelial dysfunction in Fabry disease

Background

Fabry disease (FD) is caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GLA) resulting in the accumulation of globotriaosylsphingosine (Gb3) in a variety of tissues. While GLA deficiency was always considered as the fulcrum of the disease, recent attention shifted towards studying the mechanisms through which Gb3 accumulation in vascular cells leads to endothelial dysfunction and eventually multiorgan failure. In addition to the well-described macrovascular disease, FD is also characterized by abnormalities of microvascular function, which have been demonstrated by measurements of myocardial blood flow and coronary flow reserve. To date, the relative importance of Gb3 accumulation versus GLA deficiency in causing endothelial dysfunction is not fully understood; furthermore, its differential effects on cardiac micro- and macrovascular endothelial cells are not known.

Methods and Results

In order to assess the effects of Gb3 accumulation versus GLA deficiency, human macro- and microvascular cardiac endothelial cells (ECs) were incubated with Gb3 or silenced by siRNA to GLA. Gb3 loading caused deregulation of several key endothelial pathways such as eNOS, iNOS, COX-1 and COX-2, while GLA silencing showed no effects. Cardiac microvascular ECs showed a greater susceptibility to Gb3 loading as compared to macrovascular ECs.

Conclusions

Deregulation of key endothelial pathways as observed in FD vasculopathy is likely caused by intracellular Gb3 accumulation rather than deficiency of GLA. Human microvascular ECs, as opposed to macrovascular ECs, seem to be affected earlier and more severely by Gb3 accumulation and this notion may prove fundamental for future progresses in early diagnosis and management of FD patients.     

Effect of benfotiamine on advanced glycation endproducts and markers of endothelial dysfunction and inflammation in diabetic nephropathy

Background

Formation of advanced glycation endproducts (AGEs), endothelial dysfunction, and low-grade inflammation are intermediate pathways of hyperglycemia-induced vascular complications. We investigated the effect of benfotiamine on markers of these pathways in patients with type 2 diabetes and nephropathy.

Methods

Patients with type 2 diabetes and urinary albumin excretion in the high-normal and microalbuminuric range (15–300 mg/24h) were randomized to receive benfotiamine (n = 39) or placebo (n = 43). Plasma and urinary AGEs (N ^ε-(carboxymethyl) lysine [CML], N ^ε-(Carboxyethyl) lysine [CEL], and 5-hydro-5-methylimidazolone [MG-H1]) and plasma markers of endothelial dysfunction (soluble vascular cell adhesion molecule-1 [sVCAM-1], soluble intercellular adhesion molecule-1 [sICAM-1], soluble E-selectin) and low-grade inflammation (high-sensitivity C-reactive protein [hs-CRP], serum amyloid-A [SAA], myeloperoxidase [MPO]) were measured at baseline and after 6 and 12 weeks.

Results

Compared to placebo, benfotiamine did not result in significant reductions in plasma or urinary AGEs or plasma markers of endothelial dysfunction and low-grade inflammation.

Conclusions

Benfotiamine for 12 weeks did not significantly affect intermediate pathways of hyperglycemia-induced vascular complications.

Trial Regristration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00565318","term_id":"NCT00565318"}}NCT00565318     

Antiangiogenic activity of 2-deoxy-D-glucose

Background

During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated.

Methodology/Principal Findings

In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG''s ability to interfere with endothelial N-linked glycosylation. 2-DG''s effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG''s effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH_BETAT_AG transgenic retinoblastoma model.

Conclusions/Significance

In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.     

Circulating angiogenic factors in patients with thromboangiitis obliterans

Background

Thromboangiitis obliterans (TAO, also known as Buerger''s disease) is a non-atherosclerotic inflammatory vascular disease that primarily affects arteries in the extremities of young adult smokers. Since the etiology of TAO is still unknown, therapeutic options are limited. Recent attempts in therapeutic angiogenesis have been promising. Therefore, the aim of our study was to evaluate angiogenic processes and factors including circulating progenitor cells in TAO.

Methodology/Principal Findings

TAO patients with critical limb ischemia and age- and gender-matched nonsmokers and smokers without cardiovascular disease (n = 12 in each group) were enrolled in the study. Flow cytometric analysis of peripheral blood showed significantly decreased levels of circulating CD45^dimCD34⁺ progenitor cells in TAO patients and in smokers compared to nonsmokers. In contrast to both control groups, the proportion of CD45^dimCD34⁺ progenitor cells co-expressing VEGF receptor-2 (VEGFR2) was significantly elevated in TAO patients. Enzyme-linked immunosorbent assay (ELISA) of common angiogenic factors (such as VEGF) did not clearly point to pro- or antiangiogenic conditions in serum or plasma of TAO patients. Serum of TAO patients and controls was evaluated in proliferation, migration (scratch assay) and spheroid sprouting assays using human umbilical vein endothelial cells (HUVECs). Serum of TAO patients exhibited a diminished sprouting capacity of HUVECs compared to both control groups. Proliferation and migration of endothelial cells were impaired after treatment with serum of TAO patients.

Conclusion

Levels of circulating progenitor cells were altered in TAO patients compared to healthy nonsmokers and smokers. Furthermore, serum of TAO patients exhibited an antiangiogenic activity (impaired endothelial cell sprouting, migration and proliferation) on endothelial cells, which may contribute to vascular pathology in this patient population.